CA1210811A - System for identification of cells by electrophoresis - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Cells are identified electrophoretically through isoenzyme analysis. The use of standard and control cells allows semi-skilled workers to achieve re-producible results. A coding sequence is assigned to the cells to be identified using a grid to interpret the electrophoretic separations observed on a series of elec-trophoretic films, each film being developed for a par-ticular isoenzyme. Comparison of the coding sequence with a compendium of coding sequences for known cells identifies the cells to be identified.

Description

Lepp-~ussatto-Ziegen~eyer 10-1-4 r 8~

~lOl SYSTEM FOR IDENTIFICATION OF CELLS BY ELECTROPHORESIS
.15 BACKGROUND OF THE INVENTION

l. Field of the Invention This invention relates to the identification of 5 cells by electrophoresls. More particularly, the in-v~ntion relates to a system for cell identification which can be easily used by semi-s~illed workers and yet produce consistently reproducible results.

2. Description of the Prior Art ~he identification of biological cells has been a long standing problem in the biological sciences. A
Jariety of techniques have been applied to the problem. For example, infectious microorganisms have been identified in clinical laboratorles by means of simultaneously app!ied biochemical reactions. Vertebrate, plant and mycopla~ma cells have been characterized in some research labor-atories by specilized techni$ues including sophisticated isoenzyme electrophoresis, cell surface antigen analysis and chromosome analysis.
The problem of identiIying cells is particularly acute when cells are grown in tissue culture. Because the tissue cuLture medium is d2signed to encourage cell growth, it is highl~ susceptible to contamination. As a result, a .
tissue culture thought to be growing a particular ~ype of , ,.

cell, in fact, through contamination, can be growing either a combination of the original cells and ~he contaminating cells, or, depending on the relative viability of the original cells and the contaminating cells, just the con-taminating cells. ~ccordingly, when a researcher conducts experiments and reaches conclu~ions based on tissue cul-ture work, and coupies those results and conclusions t~ a particular type of cell, he may be misleading ~oth him~elf and the public with regard to his work. Numerous ~xamples of such contamination have been doçumented in the bio-logical literature. Indeed, in 1976, it was reported in the American Journal of Hematology that approximately 30% of a series of tissue cultuxe cells studied were incorrectly designated by the subrnitting investigator. See C: S.
Stulberg, W. D. Peterson, Jr., and W. F. Simpson, "Iden-tification of Cells in Culture", American Journal of Hem-atolo~y, Volume 1, pages 237-242 tl975).
The problem of contamir.ation of tissue culture cells was identiied at least as early as the late 1960's.
~0 See, for example, S. Gartler "Apparent HeLa Cell Con tamination of Human ~eteroploid Lines," Nature, Volume 217, pages 750-751 (1968). Yet, reports of contaminated tissue cuLt~re celLs continue to appear in the literature.
See, or exampIe, D. Dickson, "Contaminated Cell Lines", Nature, Volume 289, page 227 (1981).
Of significance with regard to each af these re~orts- o~ contaminated tissue cul~ure cells i~ the rac~
.

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that the contamination was found only by means of hi~hly sophis~icated and time consuming procedures using expen-sive equipment in the hands of experts in the area of cell identification. Moreover, none ~f the prQcedures used by these experts included provisions for standardization of the system or provisions for the inciusion of a control to verify the performance of the system. Also, in order to confirm any cell identification, these investigators main-tained large stocks of reference cells. For all of these reasons, the procedures previously used for identi~ication of cells have not been reproducible enough in various in-vestigators' hands to be of general utility.

SUMMARY OF THE INVENTION
It is an object of this in~ention to provide a L5 system for the identification of cells which overcomes the problems inherent in the prior axt systems. In particular, it is an object of the inventio~ to pro~ide a cell iden-tification system which can be used generally in various laboratories and which will produce reproducible results within any one laboratory and among various laboratories.
Moreover, it is an object of the invention to provide a cell identification system which is easy to use by semi-skilled wor.kers. It is a furt~ér ob~ect-of the invention to provide a cell identification system which is inexpensive to use.

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In accordance with one aspect of the invention, an elec~rophoretic method for identifying cells is pro-vided which includes.
performing an electrophoretic separation on a S sample from each of a first, a second and a third population of cells, the firs~ p~puIa~ion being of the cells to be identified and the second and third populations being of cel1s whose identit.ies are known, each of the samples containing at least some electrophoretically sepa.rable substances which are char~cteristic of k~own cells, the separations being performed under comparable conditions;
developing the electrophoretic separations with regard to one or more electrophoretically separable sub-stances which are characteristic of known cells, the sub-lS stances.chosen,for development being Xnown to be producedby cells of both the seoond and third populations and the number of substances chosën for development being suf-ficient to characterize the cells to be identified;
comparing the developed separations for the sec-ond and third samples to determine the range of separationfor the chosen substances. under the conditions used; and ~ based on the range or separation so determined, comparing-the separations for the first and second samples to determine the identity of the cells of the first popu-lation.
I~ accordance with another aspect of the ln-`~ vention, a ~ethod for i.dentiryins cells is provided which Lncludes:

preparing an extract from a population of the cells to be identified, the extract containing subs~ances which are produced by the cells and which are electropho~
retically separable;
perfo~ming electrophoretic separations on a series of s~mples from the extract;
developing the electrophoretically separated samples with regard to a series o~ preselecte~ substances known to be electrophoretically separable and known to be produced by at least som~_ce~ls, each sample being de-veloped for one such substance and the number of substances in the ~exies being sufficient to characterize the cells to be identif.ied;
determining ~rom the developed qamples the elec-trophoretic mobility exhibited by the cells to be iden-tified for each of the preselected substances;
assigning to the electrophoretic mobility for each substance so determined: a symbolic representation;
arranging the symbolic representations in a pre-determined. order to form a codins sequence; and comparing the coding sequence with a compendium of cod.ing se~uences for known cells to determine the identity of the cells to be identified...
In accordance-with a further aspect of the in-ven~ion, a ~ethod or electrophoretically identifying celLs is provided which inclu~es:
selecting a series af electrophoretically.sep arable substances, the number o~ substances in the series being sufficient to permit characterization of the cells to be identified; .' performing ~ fi~s~~set of electrophoretic sepa-rations on extracts from a plurality of known celis, the separations being performed under conditions which limit the differences in distances migrated for the series of substances and for the pluxality of ~nown cells to a derined range corresponding to a predetermined scale for inter-preting the separations;
developi~g the first set of separations ror the series of electrophoretically separable substances;
interpreting the first set of developed separa-tions u~i~g the predetexmined scale so as to assign a sequencing code to each of the known cells;
lS performing ~ second set of electrophoretic sepa-rations on extracts from the cells to be identified under the conditions used for the known cells;
developing the second set of separations for the series of electrophoretically separable substances;
interpreting the second set of developed separa-tions using the predetermined scale so as to assisn a sequencing code to the cells to be identified; and comparing the sequencing code for the ceLls to be identified with the sequencing codes f`or the kncwn cells to determine the identity of the cells to be identified.
In accordance with a still further aspect of the invention, a method for identifying cells is provided which~ ~-includes:

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electrophoretically separating extracts from standard oells, control c lls and ~he cells to be iden-~iied on separate tracks of an electrophoretic film;
developing tne electrophoretic film with regard to a first preselected electrophoretically separable sub-stance using a reagent specific for that substance to produce a pa~tern of one or more bands of an insolubl~
product in each track for which the substance is present in the extract, the pattern, where present, being indicative of the extent of electrophoretic migration of the first preselected substance fot the standard and control cells and for the cells to be identified;
interpreting the developed electrophoretic film by reference to a grid having a plurality of spaced lines thereon and having a sequence of symbols associated with the lines, the interpretation ~ccurring by aligning the film with the grid by reference to the developed pattern in the track for the standard cells and determining by ref-erence to the grid~first and second symbols corresponding to the developed patterns in the tracks for the extracts from the control cells and the cells to be identified, re-spectiveLy;
recording the first and secon~ symbtals so de-; termined at first an~ second locations, respectlvely, on a data sheet;repeatlng th~ separatingt developing, inter-preting and recording steps for a predeterminèd number of ~2~S~8~

additional electrophoretically separable substances suf-ficient to characterize the cells to be identified, the symbols determined for ~hese substances being recorded on the data sheet at predetermined locations located relative S to the first and second locations to form a flrst coding se-quence for the control cells and a second coding sequence for the cells to be identified;
comparlng the coding sequence for th~ control cells with a rererence coding sequence for those cells to l~ determine whether the electrophoretic separations have been performed properly; and for properly performed separations, comparing ~he coding sequence for the cells to be identified with a compendium of coding sequences for known cells to determine the iden~ity of the ceLls to be identified.
In accordance with a still further aspect of the invention, a method for identifying cells is provided which includes preparing a plurality of extracts from the cells to be identified, the extracts containing electropho-retical.ly separable isoenzymes, electrophoretically sep-arating the extracts and developing the separated extracts with regard to the isoenzymes purine nucleoside phos-phorylase, glucose-6-phasphate dehydrogenase, malate de-hydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase- and lactate dehydrogenase to produce the same insoluble product for each of the iso-enzymes.

In accordance with a still further aspect of the invention, a sys~em ~or identifying cells elec~rophoretic-ally i5 provided comprisingo - an extract fron~ standard cells;
an extract from control cells;
a set of reagen-~s for developing electrophoreti~
separations, each reagent being identified by a code dis-tinct from the codes used for each other reagent, the number o reagents being chosen to permit characterization of the cells to be identified;
a set of electrophoretic films having-means for receivi~g samples from the extracts of the standard and control c~Ils and from an extract prepared from the cells to be identified, the films being identified by the ccdes used for the reagents, one code for each film; and means for interpreting the el~ctrophoretic films including means for compiling a sequence of symbols in-dicative of the separation achieved for the cells to be identified for the set of reagents.
. In accordance with.a stifl further aspect of the invention, a grid is provided Eor use in interpreting electrophoretic separa~tions of extracts from cells, the separation ~eing performed a~ an electrophoretic film and the:film ha~ing a plurality af~tracks includin~ a track for separation o~ an extract from standar~ cells and at least one-trac~ for separation of an extract from cells to be identified~, which comprises a plura~lity of spaced lines, one or the lines serving to position the film ~ith respect :
_g ~2~

.
to the grid by reference to the separation in the track con-taining the extract from the standard cells and the re-maining lines bein~ spaced relative to the positioni~g line and relative to each other for discriminati.ng differences in electrophoretic mobility between the sta:ndard cells and each of a plurality of known cells with regard to a series of preselected electrophoretically separa~le ~ubstances, the number of substances in the series being chosen to permit characterization o~ the cells to be identiied.
The methods and apparatus of the invention are specifically adapted. to identify cells by means of iso-enzyme analysis. Among the isoenzymes for which analyse~
are preferably performed are purine nucleoside phosphory-lase, glucose-6-phosphate dehydrogenase, malate dehydro-genase, mannose phosphate isomerase, peptidase B, aspar-tate aminotransferase and lactate dehydrogenase. Amon~
the cells which preferably can be used for ~he standard cells are mouse L cells. Among the cells which can be used for the control cells, are HeLa cells. Among the symbols which preferably can be use~ to form coding sequences indicative of the various~ cells are letters of the. al-phabet. Amons the ways the various reagents preferabLy can be ldentified with a d.istinctive cod~ is through color-codln~, The attalnment of the above objects of the:in-vention as we-ll as other ob~ects is described below in connection with the description of the preferred embodi-ments O

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BRIEF VESCRIPTION OF THE DRAWIN~

Figure 1 is a perspec~ive view s,howing ~he re-agents and the extracts from the standard cells and the control cells as supplied to the user in accordance with 2 preferred embodiment of the invention.
Figure 2 i5 a perspective view showing the ap-plication of a sample of a cell extract -to an electro-phoretic film.
Figure 3 is a plan view of a grid for use in interpreting electrophoretic separations in accordance with the invention.
Figure 4 is a side view of the grid of Figuxe 3 taken along line 4-4 in Figure 3.
Figure 5 is a plan view of the grid o~. Figure 3 with an electrophoretic film in place for interpretation.
Figure 6 is a pla~ view of a data sheet ~or use in compiling sequencing codes to identify cells in ac-cordance with the invention. Figure 6 also ilLustrates storage of electrophoretic films in a pocket which forms part of the da~a sheet.
Figure 7 illustrates one page of a compendium o~
sequencing codes for use in identifying cells i~I accoxdance with the invention.
Figure 8 is a plan vie~ of ~n electrophoretic ~5 film supported on a plastic holder. As shown in this figure, the film lies below the holder.

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Figure ~ is a side view of the elec~rophoreti~
film and holder shown in Figure 8.
Figure 10 illustrates a series of seven films . developed for a series of seven electrophoretically sep-arable substances. This figure, in oombination with Fig-ures l and 6~ illustrates the color coding of the reagent containers, data sheet and electrophoretic films with regard to ~he various reagents as indicated by khe shadings and cross-hatchings used on the labels on the containers., 1~ the labels on the films, and below the data entry locations on the data sheet.
Figure 11 is a representative calibration curve for determining total enzyme activiky, in this case, for the enzyme peptidase B.

DESCRIPTIO~ OF THE P~EFERRED EMBODIMENTS

The present invention provides a systematic way by which semi-skilled worXers can successfully perform so~histicated electrophoretic analyses to determine the identity of cells. The user is provlded with premeasured reagents or developin~ electrophoretic films and with standardized extracts from both standard cells and control cells. Fur~her, he is pravided ~ith a specialized grid for interpret.ing electrophoretic films and.a data sheet both for recording the Lnterpretakions as they axe made and for - 25 compiIin~ a sequencing cod~ Lndi~ativë~of the identity of the cells to be ide~tified. Pinally, the user is sup~lied with a compendium of sequencing codes for previousLy iden-~ 2~C3~

tified cells so that a comparison can be made between the sequencir,g code compiled for the cells to be identified and the previously determined sequencing codes to determine the identity of the cells t~ be identified. By using the components of the system in the manner described below, ceLl identifications can be performed routinely by persons having little or no formal traininq ~n the field of electro-phoretic analysis.
In the discussion that follows, specifio ex--10 amples o~ the use of the method and apparatus of theinvention to perform isoenzyme analysis are given. In particular, th~ invention is illustrated for the se~en isoenzyme.s- -- purine ~ucleoside phosphorylase t~P), glucose-6-phosphate (G6PD), malate dehydrogenase tMD), mannose phosphate isomerase (MPI), peptidase B (Pep B), aspartate aminotransferase (AST) and lactate dehydrogPnase (LD) -- and for mouse L cells as the s~andard cells and ~eLa cells as the control c lls.
I~ is to be understood that,,the invention is not ~o limited to isoenzyme analy~is7-nor to the seven isoenzymes illustrated~ but can be used'with any electrophoretically separable substance which ca~ be extracted fxom biolog-ical cells, such as proteins r and in particular, any set of isoenzymes, ha~in~ more or lessithan seve~ members. Also, well-characterized cells other than mouse L and HeLa can be used as the-standaxd and contol cells, respectively. ~mong the cells; which fall within this categary are Chinese hampster ovary cell~, mouse liver cells, mouse kidney cells, veal kidney cells, and the like. ~hese cells, as well as mouse L and ~eLa cells, can bP used as either the control or standard ceils.
S The number of electrophoretically separable sub-stances for which analyses are performed will vary with the nature of the cells to be identified. For example, if the cell5 to be identified are otherwise well-characterized, only a few, or even just one, electrophoretically separable substance need be analyzed. In other cases, more than the seven analyses illustrated herein may be necessary to identi~y the cells under study. In ge'neraL, the number of substances is chosen to p~ovi~é a reaconable assurance that the identificati~n attributed to the cells to be identified is correct.
Although the invention is described in terms of analyzing extracts ~rom populations of ceils grown in tissue culture, it is to be understood that the invention is not limited to cell extracts rom such sources. Rather, the invention can be used with cell extracts from any source, prcvided the extract contains electrophoretically separable su~stances. Thus~ ~he invention can be practiced directLy on cell~ taken rom~ among other things, human or animal tumors, ~ithout the need to grow the cells in tissue 2S culture. Also, ~he inventiQn can be applied t~ other fluids containing-electrophoreticalLy separable~substances, such as blood, lymph, urine, sali~a and ~he like, as well as to synthe~l materials.

. In practice, the materials needed to use the invention are preferably provided in the form of a kit or system having all of the specialized items required to identify cells in accordance with the in~ention. Figures l-ll illustrate the components of su~h a kit.
Figure l shows---t~e reagent ancl standard and control ceLl extract portions of the kit. The reagents are contained in reagent containers 22. Four color-coded sets of containers 22 are provided: two sets for developing electrophoretic films and two sets for per~orming total en~yme anaLyses~ Each container holds a premeasured amount of reagent ~or either developing an electrophoretic film or for performing a total enzyme analysis. Whe~ used for development~ the premeasured reagents are dissolved in l.0 milliliter of barbital buffer, pH 8.6; when used for total enzyme analysis, the reagents are dissolved in 5.0 mil-liliters of the same buffer. The bu~fer optionally is provided with the kit.
As shown in Fisure l, the four reagent containers 22, ~or any particular reagent, are arranged in a single column i~ carrying tray 24. Tray 24 includes apertures 26 for receiving containers 2Z~ It can be made of any suitable ~aterial, such as styrofoam.
In Figure 1, for pur~oses of illustration, the reagent containers 22 have been arranged from left.ta right in -tAe order purine nucleoside phosphorylase (NP), glucose-6-phosphate dehydrogenase (G6PD), malate dehydro-genase (MD), mannose phosphate isomerase (MPI), peptida e B (Pep B), glutamate oxalace~ate transaminase, also known as aspartate aminotransferase ~AST), and lactate dehydro-genase (LD). Each o~ the reagent containers 22 carries on its label 28 the abbreviation for the isoenzyme (e.g. NP, G6PD, MD, MPI, Pep B, AST and LD), as well as a number between 1 and 7 for the isoenzyme ~e.g. 1 for NP, 2 for G6P~,

3 for MD, 4 for MPI, 5 for Pep B, ~ for A5T and 7 for LD).
The abbreviations and numbers also are printed on label 30, on txay 24, below ~ach column. Further, each of the labels 2a, as we.lL as label 30 on tray 24, is colored-coded for the isoenzymes (e.g. yell~ for NP, orange for ~6PD, magenta for MD, purple for MPI, blue for Pep B, dark green for AST
and light green for LD). The hues, tints and intensities of these colors are chosen so that e~en color-blind users will perceive some difference among the various labels. By use af this three tier coding approach -abbreviations, numbers and colors--the possibility that unskilled~workers will mix up the reagents is reduced to a minimum.
The four sets of reagents are provided so that one kit can be used to make two complete sets of analyses.
As discussed above, each complete analysis includes elec trophoretic separations and totaL enzyme analyses. This consumes the contents of two reagent- containers 22 from each column.

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Tray 24, in addition to carrying reagent con-tainers 22~ also carries c~ntainers 32 and 34 which hold extracts from two standardized cells. As discussed below, one o. these extracts ls used as a standard ~.container 32) and the other as a control (container 34). In a preferred embodiment of ths i.nvention, mouse L cells a~e used as the standard and HeLa cells are used as the control. Containers 32 and 34 are held in apertures 36 in tray 24; they carry "STANDARD" and "CONTROL" labeLs 38 and 40, respectivelyG
Because the reagents and extracts are heat sen-sitive, tray 2a and its contents~preferably are stored in a refriqeratar at 2 -8 C.
The standard and.control extracts are supplied in freeze-dried. form. Prior to use, the extracts are reconstituted in containers 32 and 34 with 200 microliters o~ a buffer composed of Tri~ (tris(hydroxymethyl)amino-methane), p~ 7.5, 50% glycerine and O.l millimolar EDTA
(ethylenediaminetetraacetic acid~. Once reconstituted, the extract can be stored for extended periods of time at ~20 C.
In addition to the reagents an~ the extracts, the kit supplied to users includes a set of electrophoretic rilms La (Figure 2). A total of sixteen films normally are provided wit~ each kit. Because eac~ analysis requires seven films -- one film for each of the .~even isoenzym2s -- and because each kit ha~ enough reagent for two complete analyses, the sixteen films are actually two more than what is needed. The two extra films are included to provide one - . .. - -8~L

extra film for each analysis in case a film is accidentally harmed during handiing.
Electrophoretic film 10 is composed of a support 16 upon which is coa~ed an elec~rophoretio medium 18 (Figure 2)~ Support 16 can be made of a variety of materials including polystyrene and polyethylene tereph-*

thalate (e.gO Mylar)~ A preferred material is polyst:yrene.El~ctrophoretic medium 1~ can consist of agar, agarose, starch, cellulose ace~ate, polyacrylamide, or the like, in a buffer. ~ prefer.red electrophoretic medium 18 i5 com-posed o~ l~ (w/v~ agarose, 5% (w/v) sucrose and 0.035~ (w/v) EDTA disodium salt in a 0.065M barbital buffer, p.R. 8.6.
A medium of this general type i5 described in U.S. Patent 3,766,047.
15To facilitate handling of film lO, the ilm is supplied to the user on a plasti holder 14 (Figures 8 and 9). Support portion 16 of the film extends beyond holder : 14 at 42 which allows the film to be peeled back and removed from the holder. Electrophor~tic medium 18 is sandwiched between holder I4 and support 16 and thus is protected durins shipping and handling. Holder 14 includes ports 44 which are used to apply electrophoretic medium 18 to support 16 after support 16 ~nd holder 14 have been combi~ed as a unit. The details of th~ me~hod for applying the medium 18 to support 16, which method forms no part of the present invention, are described in U.S. Patents 3,47g,265 and 3,767,560.
* trade mark Electrophoretic medium 18 includes four sample wells 12. These wells define a set of four lanes, numbered 1, 2, 3 and 4 in Figure 8. As shown in F:igure 2~ samples are applied to the medium by hand, using a miGrOpipette 46.
One sampIe is placed in each of welLs 12. The composition of the samples applied to the individual wells is described below.
Included in the kit are a set of labels 20 for the electrophoretic films 10 ~see Figure 10). These labels carry the same three~tier coding (abbreviation, number and color) u~ed ~or reagent containers 22 and label 30 on tray 24. A total of 14 labels are supplied with each kit, that is, two sets of seven labels, each of the seven being coded for a particular isoenzyme. For each complete analysis, one label 20 from each set of seven labels is applied to each of seven electrophoretic films 10 prior to removal of the films from their holders 14. This gives the user a set of se~e~ coded electrophoretic films 10 for use in making one complete electroph~Eetic analysis for the seven i50-enzymes for which reagents are supplied in tray 24. Labels20 are supplied with a suitable adhesive on their front faces for attachment to the rear faces of support portions 16 of eLectro~hoxetic films 10, i.e~, the aces o~ su~ports 16 which do not carry electrophoretic medium 18 (see Figure 10).
- Labels 2Q, in addition to being coded for the isoenzyme reagents., are provided wi.th lane designations, specirically, the designations "standard", "control'l, "sample 1", and "sample 2'l. When labels 20 are attached to electrophoretic films 10, th~se ~esignations line tlp with lanes 1, 2, 3 and 4, respectively, on films 10. The designations show the user which samples to place in each of sample wells 12. Thus, using micropipette 16, the user places a sam~le from container 32 (the standard extract container) in well 12 in lane 1, designa.ed "s~andard" on label 20. Using a clean micropipette, the user then places a sample from container 34 (the control extract container) in well 12 in lane 2, designated ",control" on label 20.
Finally, again using cl~an-mLcropipettes, the usex plaes a sample from each of up to two cell extracts to be identifi.ed in the sample wells 12 of lanes 3 and 4, lS corresponding to the designations "sample 1" and "sample 2". One microliter samples can be used. This process is repeated for each of the seven electrophoretic fil~s 10 to which a label 20 has been applied. Note that a total of four cell populations ,~an be analyzed with one kit; each kit includes enough supLies for two complete analyses, and each analysis can accep,t samples from two cells to be lden-tiied. ~he two sets o~ reagents for use in determining total enæyme, activity similarly are sufficient to perform two anaLyses each for-each set.
In addition to the reage~ts and the films, the kit also includes a gria 48 for interpretinq electro ~horetic films 10 (Figures 3 and 4). Grid 48 includes body 50 and alignment bumper 52 which projects above body 50 at the left-hand side of grid 48. Body 50 carries thereon a plurality of spaced lines 54 having associated therewith letters of the alphabet 56. Body 50,also includes a thick line 58 which is desig~ated-"STANDARD".
As is discussed in detail below, after samples have ~een placed in sample wells 12 for the complete series of seven electrophoretic films lO, the films are subjected to electrophoretio separation on electrophoretic sepa-L0 ration apparatus. Next, they are developed with thereayents contair.ed in reagent containers 22, one reagent being used for each filn7., with the three tier coding on film labels 20 and reagent labels 28 being used to select the appropriate reagent for each film. This process ca~lses coLored bands 60 to appear in the lanes of electrophoretic film I0. Normally, the reagents and the standard and control cells are chosen so that at least one band occurs in each of lane7s 1 and 2. Whether bands appear in lanes 3 and 4 for samples l and 2 depends on the characteristics of the cells ts be identified, although normally the analysis is structured so that most cells produce a band7 The locations of th~ bands in lanes 3 and 4 are indictative of the cells to be identified Grid 48 is used as the fir3.
step in interpreting these locations so as to arrive ultimately a-t a~ identi~ication of the cells to ~e iden-t~fied.

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The use of grid 4% is illustrated in Figure S.
As shown therein, an electrophoretic film 10 i5 aligned witn the grid 48 in two ways -- firstr by sliding the film against alignment bumper 5Z, and second, by placing band 60 5 in the lane designated "standard" over thick line S8! i.e., the "stand~rd" line. Once this alignment has been made, the user reads off a letter of the alphabet 56 (or letters of the alphabet, i~ approprLate), corresponding to the lo-cation of band 60 in each of the lanes designated "control", "sample 1" and "sample 2". For example, in Figure 5, the letters ~or these three lanes are H, K and F, respectively.
It should be noted that although letters of the alpha~et 56 have been used in Figures 3 and 5~ other symbolic rep-resentations, e.g., numbers, could be used equally as well.
Figure 5 shows grid 48 being used for an elec-trophoretic film 10 carrying a label 20 designating purine nucleoside phosphorylase, NP. The same interpretatlon process is carried out for each of the other films in the series. That is, each film is aligned with bumper 52 and thick line 58, and.a Letter (or letters) of the alphabet 56 is assigned to the location of the bands in the "control"
lane and tv the bands, if present~ in the "sample 1" and "sample 2" lanes. Accordingly, when the proce~s has been completed ~or all seven films, the user will have, in the usual case~ three series of seven lettersr one series for ` each of the control, sample l and sample 2. To keep track o this data and the steps of the interpretation, the kit includes data sheet 62 (Fiqure 6).

-Data sheet 62, in the embodiment shown in Figure 6, includes four rows 64, 66, 68 and 70 and nine columns 72, 74, 76, 78, 80, 82, 84, 86 and 88. It also includes pocket 90 for holding developed electrophoretic films 10 and a portion 92 for recording comments. The data sheet is designed to fold about line g4 to provide a convenient pac~age for storing cell identification information~
The four rows 64, 66, 68 and 70 of data sheet 62 correspond to the "standard", "control", "sample 1" and 1~ "sample 2" lanes of electrophoretic films 10. Columns 74 through 86 correspond to the seven isoenzymes for which electrophoretic films L0 have been developed. ~hese col-umns carry the same three tier coding--abbreviations, numbers and colors--as used to co~e film labels 20 and reagent container labels 28. Column 72 identifies rows 64, 66, 68 and 70 as relating to the "standard", "control", "sample i" and "sample 2". Column 88 is used to record the uLtimate identification or the cells to be identified, the ~rocess for which is described below.
Data sheet 62 is used in conjunction with grid 48 by recording in the appropriate column and row the letters 56 determined for each Lane of each electrophoretic film 10. Thus~ for exampl~e, ror the NP film shown in Figure 5, an H is recorded in column 74, row 66, corresponding to the band at levei ~ in th~ "control" lane (lane number ~) of electrophoretic rilm 1~. Similarly, for lane 3, a R is recorded in columQ 74, row 68, and for lane 4, an F is ..

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recorded in column 74, row 70. All the recordings are made in column 74, because this column corresponds to ~he NP
electrophoretic film shown in Figure 5. The three tier coding serves to minimiæe transposing data for the seven s isoenzymes. Row 64 of data sheet 62 includes a check-off box entitled "film aligned" to serve as ~ reminder that the letters 36 are to be read only after the film has been aligned with bumper 52 and thick Line 58.
The process used for the NP electrophoretic film 10 is repeated for the remaining six electrophoretic ~ilms in the serLes, each letter 56 being entered in its appro-priate space undex the guidance o~ the three tier code and the cell identifications in column 72~ Upon completion of these steps, which can be performed by persons with very little experience in electrophoretic analysis, there will exist on data sheet 62 a compiled coding sequence o~ letters 56 corresponding to the control cells ~row 66~, the sample 1 cells (row 6aJ and the sample 2 cells trow 70). For example t in Figure 6, the coding sequence for the control is HIKIIOO, for sample one, KJJJKOK and for sample 2, FIIJ~UG. With these coding sequences in hand, the final step of the identification is pexformed: comparing the compiled c~ding sequences~ with known coding sequences to determin~ the identity ~f the cel-ls to be identified. This is done~by means of a compendium o~ coding sequences. Fig-ure-7 ill,ustrates a page, identified by the ~umber 36, of such a compendium prepared' in printed book form.

.

The compendium is compiled hy conducting elec-trophoretic separations on numerous known cells and inter-preting thcse separations using grid 48 and data sheet 62 to compile a sequencing code for each of the known cells.
The separations are run under the conditions to be used in identifying cells to be identified. A de~aile~ discussion of these conditions i~ presented below in connection with the description of how extracts are prepared and electro-phoretic separations are performed. Brie~ly, one set of variables which preferably are kept constant are the sizes, types and thicknesses of the electrophoretic media 18 of electrophore~ic films L0. ~ther variables include the time, rield strength and temperatures at which the elec-trophoretic separations are performed.
As explained below, greater-varia~ility can be tolerated in the separation and development conditions because the pattern for the cells to be identified is com-. _ ,, _ .
pared with ~he pattern ~or the standard cells. Since these 'wo patterns are created and developed on the same electro-phoretic film 10, discrepancies from the prescribed conditions tend to effect both patterns ~ore or less equally, so that the differences between the patterns, ~hich is what is measure~ to determin~- th~- identity of the cells to be identified, remains generally ~onstant. And, as also explained below, by uslng an extract of contr~l cells, agaLn on the same electrophoretic film 10, a ready check is provided to determine if the limits of variability have been exceeded.
Once the sequencing codes for various known cells have been determined, the codes are arranged in the compendium in any convenient order. The compendium can be in the form of a printed book as illustrated in Figure 7 or can be in the form of a data base for a computer, micro-processor, or the like~ These la~ter forms for the com-pendium are preferable for analyses based on large numbe:rs of isoenzymes, e.g. f~rty or fiîty isoenzymes, rather than seven as used for illustration herein, and for large compilations of sequencing codes for known cells.
A~ter the initial round of sequencing code de-terminations for known cells has been made and placed in the compendium, additional entries can be made as more known cells are studied and their sequencing codes determined.
The compendium, whether in book form or otherwise, can be supplied ~ith each kit or as a separate item. Updated editions of the compendium als~ can be issued as sequencing codes for more cells are determined.
Using the compendium, the ultimate identifi-cation o~ the cells to be identified is made and entered into column ~8 of data sheet 62. This is done by first comparing the sequencing code ~or the control cells ~row 66) with the compendium. I the electrophoretic sepa-rations and development ha~e been done properly~ the se-; ~uencin~ code for the control should agree with the se-quencing code given in the compendium for the control . . _ .

~21~8~

cells. Thus, for our illustrative example of He~a control cells, the coding sequence which appears in row 66 should be the same as the sequence which appears in the compendium for HeLa cells. This is an important check on the opera~ion of the system and is one of the aspects of the invention that allows sophisticated electrophoretic analyses to be performed by semi-skilled workers. By receiving fee~back in the form of a match between the sequencing cvde deter-mined for the control cells and the known sequencing code for those cells, the workex Xnows that he has performed the anaLysis properly. This yives the worker confidence in his results, and allows others to rely on his worX. Also, if a match i5 not achieved, the wor~er immediately Xnows that the compendium should not be use~ in interpreting the coding sequences for samples 1 and 2 (rows 68 and 70 respectively). Other techniques of interpretation, dis-cussed below, may then be appropriate.
Assuming there is a match between the sequencing code in row 66 for the control cells and the known se-quencing code for those cell.5, the worker then consults thecompendium for sequencing codes corresponding to the se-~uencing codes compiLed for samp}e~ 1 and 2 (rows 68 and 70 respectively). I~ matches are foun~, the cell identi-fications associated with the code~ in the compendium are ..
entered in column ~8 of d~t-~ sheet 62, thus completing the analysis~ For example, in Figure 6, the sample l and sample 2 sequencing codes correspond to the sequencins codes for Syrian hampster and dog in Figure 7. If the coding sequence is not found, it may be appropriate to report the new coding sequence to the compiler of the compendium and ~o in-vestigate the possibilLty of identifying the cells to be identifi~d by non-elec~rophoretic methods so as to ~e able to update the compendium with an additional sequencing code.
As described above, grid 48 includes a series of spaced lines 54 ha~ing associated therewith symbols 56 L0 which make up the sequencing codes used to identify cells.
Although the lines are shown equally spaced in Figures 3 and 5, it is to be understood that the lines do not have to be so spaced. Also, other forms of grids, such as linear or circular sli~ing grids can be used, provided they result in lS assigning a symbolic representation to the relative spac-ing between electrophoretically separated and developed bands of standard cells and other types of cellsO
In general terms, the speci'fic grid 48 shown in Figures 3 and 5 and other~types of grids are designed in view of the series oE electrophoretically separable sub-stance~ subject to analysis, the conditions used for the electrophoretic separations, and the nature of the stand-ard cells which are used. That is, the grid is chosen- so that by aligning the grid based on the ~and ~roduced for the standard c~llsr symbolic representations can be assigned to numerous ceLls to be identifiQd for a ser-ies of pre-selected- substances. For the seven isoenzymes used Eor 8~L~

illustration herei~, mouse L cells as the standard, and the electrophoretic separation equipment and conditions de-scribed below, it has been found that a grid of twenty equally spaced lines, ~ millime~ers apart, can be used to S assign symbolic: repxesentations to the electrophoretic bands observed for the seven isoenzymes for a plurality of cells presently grown in tissu culture includin`g r.at, hamster, rabbitr mink, monkey, cow, dog~ marmoset, gibbon and chimpanzee cells. For other isoen2ymes, standard L0 cells, or electrophoretic equipment or procedures, similar grids can be.~eveloped.
~s mentioned-abovë, it may happen that the coding sequence com~iled for the control cells is not in agreement with the known codi.ng sequence for those cells. In such a case, the separation data may still be salvageabLe. For ex-ample, the coding sequences may be different with respect to only one isoenzyme-, and the coding se~uences Eor the unknown samples I and 2 may be interpretable, with reason-able confidence,. without reference to ~hat isoenzyme.
Alternati.vely, a correction m~y be ma~e to the symbols assigned to the csll.s to be identified ~or the errant isoenzyme,-based on the difference between what the symbol ~or the control.ceLls should ha~ebeen.and wh~ was actualLy observed..
I~ practi.ce, the ki~: is use~ in the following m~nner. Firstt tw~ ceLl extracts are prepared which contain electrophoret.ically se~arable substances from the ~2~

cells to be identified (sample l and sample 2)~ The extracts can be prepared in a number of ways known to the art. One such way involves disrupting the cells byfreezing and centrifuging the disrupted cells to produce the desired extract in the form of the supernatant liquid which contains electrophoretically separable substances.
Once the ex~racts have been prepared, samples from each extract are in~roduced into sample wells 12 in lanes 3 and 4, respectiveLy, of seven electrophoretic films 10. Samples from the standard and con~rol extra~ts sup-plied with the kit are introduced into lanes l and 2, respectively, of each film. The seven electrophoretic films 10 have previously had applied 'hereto labels 20~ as described above.
Once all the samples have been deposited in the sample wells 12 of the series of electrophoretic films 10, each film is subjec~ed to electrophoretic separation. The electrophoretic separation can be accomplished with stand-ard electrophoretic separation apparatus known in the zrt.
In the case of isoenzyme analysis, the electrophoretic separa~ion apparatus ~hould include means for preventing an increase in the temperature of electrophoretic film 10 during the electrophoretic separation process so as to avoid the inactivation of the isoenzymes. I~ particular, for ~he seven iso~nzymes used illustratively herein, ithas been found that the temperature of the film should be main-tained between 4 C and 10-C throughout the separation.

An electrophoretic separation appa~atus for per~oxming the separat~on and ma~ntaining the temperature o~ the film within the preferred range was used, and it was detenmined that satisfactory separ~tions could b~ achieved for ~he seven isoenzymes for a variety of cell types usi~g thin film agarose electrophoretio films havins a polystyrene support and composed of 1% ~w/v) agarose, 5% (w/v~ sucrose and 0.035~ (w/v) EDTA dieodium s~lt in a 0.065M barbital buffer, p.H~ 8.6 (Corning ~edical Catalog ~470100); an electrode buffer of ~arbital, pH 8.6; 500 milliliters o~
tap water, 4-C to 8'C, as the cooliny medium in ~he separation apparatus; an applied field strength of 27 V/cm achieved by placing a steady 160 volts acro s the elec- .

trodes of the separation apparatus; and conducting the separation for 25 minutes.
Moreover, by p~rforming the separations in this manner, it was foun~ that the developed bands or all of the isoenzymes and for a Large variety o~ cells could be i.nter-preted using the linear grid showD in Figures 3 and 5, witha spacing of 4 millimeters between lines and a totai of twenty lines, and with mouse L cells used as the standard.
~mong the speci@s which were found interpretable with grid :

~ 8~
48 under the above conaitions were ra~, hamster, rabbit, mink, monkey, cow, dog, marmoset, gibbon and chimpanzee.
Once the electrophoretic separation has pro-ceeded for the prescribed period of kime, ~he apparatus i5 shut of~ and the electrophoret.ic films 10 removed. Reagent from one set of re gent containers 22 is then dissolved in barbital buffer, as described a~ove, and spread evenIy over electrophoretic media 18, one reage~t container being used for each film 10, the reagent containers and the films beins matched by means o~ the three.tier coding system described above. Excess reagent is rolled off each electrophoretlc film 10 with a pipette or stirring rod used sideways. Films 10 are then placed in a moist chamber at ~7 C and incubated for 20 minutes in the dark. ~ext, the films are removed and lS washed twice in deionized water at 10 minute intervals. In the case of agarose based films, the washing step is followed by air drying at approximately 60 C which causes the agarose to collapse anto and band with the support portion 16 or e-lectrophoretic films 10. This completes the separation and developing processes or electrophoretlc films lOr after which t~e fiIms are interpreted as de-~cribed above using grid 48, dat~ sheet 62 and the com-pendium of sequencing cGdes~ for known celLs.
Reagents which have been found preferable for developing the seven isoenzymes used herein to illustrate the invention have the following com~ositions:

~Z~LQ~
Purine nucleoside phosphorylase: inosine, 13.41 mg xanthine oxidase, 1 unit MTTt 7.0 mg PMS, 0~06 mg EDTANa2 0.186 mg sodium salicylate, a . 08 mg K~ PO~ 49.98 mg K2~P0~ 30.21 mg Glucose 6-phosphate dehydrogenase: NADP 1.97 mg-MgCl 6.1 mg G-6-~Na 7.1 mg ~TT 5.0 mg PMS 0.06 mg oxidized glutathione 3.15 mg Malate dehydrogenase: N~D 1~.9 mg sodium malate 31.2 mg MT'r 5.0 mg PMS 0.06 mg EDTANa O.OOS mg oxidiz~d glutathione 3.15 m~
Mannose phosphate isomerase: mannose-6-phosphate lS.~ my glucose phosphate 20 units isomerase glucose-6-phosphate 20 units dehydrogenase NAD 6.63 mg MgC12 ~H2 6.1 ~5 ~TT 5.0 mg PMS 0.06 mg citrate buffer 1.42 mg ovalbumi~ 1.0 mg oxidized glutathione 3~15 mg Peptid~se B: ~-Leuglygly 30.66 mg L-amino acid oxidase 0.1 unit MTT 5.17 mg :
PMS 0.06 mg manganese C12 4~94 mg man~itol 50.00 mg ~spartate aminotrans~erase: L-aspartic acid 7.75 mg 2-oxoglutaric acid 1.46 mg NA~. I3.26 mg g~utamic acid S0 units -dehydrogenase MTT 5.0 mg dia~horase 20 units pyrid~xal~5-phosphate 0.1 mg adenosine diphosphate 4.62 mg fla~ine- mononucLeotide 0.05 mg ~2~8~

Lactat~ dehydrogenase: L lithium lactate 14.4 mg NAD 19.9 mg MTT 5.0 ~g P~IS 0.06 mg oxidi:zed glutathione 3015 mg where NAD is. nicotinamide adenine dinucleotide; NADP i nicotinamide adenine dinucleotide phosphate; P~S i5 phena-zine methosulfate; ~TT is 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-'cetrazoli.um-bromiae. The CQmponentS for t~e Peptidase B reagent are airectly placed into reagent con~ainers 22. The components of the remaining reagents are combined with 1 milliliter water, freeæed dried an~ the resulting powder placed in containers 22. Prior to freeze drying, the p~ o these reagents is.adjusted with HCl to the lS following values: NP-pH 6.2; G6PD-pH 2.0; MD-pH 4.0; ~PI-pH 6.0; AST-pH 6.0; LD-pH 4Ø To use the dry reagents; the user adds either l.or ~ milliliters of barbital buffer, pH
8.6, depending on whether an electrophoretic or total enzyme analysis: is to be performed, respectively.
Each of thesé:rea~en~s uses the transformation of a soluble tetrazolium dye to an insoluble formazan to localize its respecti~e isoenzyme. Each formazan used is colored either purple-blue ar purple-red. Accordingly, aI.l o~ ~he films, as well as aLL o the total isoenzyme analyses,. will hav~ si~ilar coLors. This is o~ b nefit in making: the- system easy ~Q use~ by semi-skilled. workers ~ecause i~ tends t~ giv~- a sense o~ uni~ormity to the . . :

~2~81~

overall process. Tetrazoliums producing other colors, such as yellows ox reds, can also be used, as well as other insoluble read-out ma~erials having a single family of colors for the various reagents.
S As discussed above, four sets ~f r~agent con-tainers 22 are incLuded in each kit: two for conducting electrophoretic separations and two for to~al enzyme ac-tivity determinations. Total enzyme activity is deter-mined using these reagents by dissolving the dry reagents in S milliliters of barbital buffer and reacting 100 microliters of the dissoIved reagents with 10 microliter s~mples o~ the extracts from~th~ cells to be identified.
The reaction is allowed to proceed for 10 minutes after which it is stopped by adding I ml of a stopping reagent (0.05M ~Cl + 1~ Triton X-100 in deioni~ed water). The absorbance of the reaction mixture is then read ag~inst a reagent blank on a spectrophotometer set at 565 nm. The optical densi~y so measured is then converted to activity units using a calibration curveO On such calibration curve ~Q for peptidase B is shown in Figure 11. This curve was obtained using 10 microliter samples of th~ reagent for peptidase B, amounts of L leucine varying from 10 to 400 nanomoles and 1~0 ~L of the stopping rea~ent. The changes in optical de~sity at 565 n~ were ~easured against reagent blanks for rhe various amounts of I-leucine, and the curve shown in Figure lI was ~erived by defining one activity unit as the am~unt of peptidase B~which forms ane micromole o ~2~Q~

L-leucine/minute at 37 C~ Similar curves are obtained ln the same manner for the other reagents using varying amounts of the product produced by the reaction of the isoenzyme with its reagent~
The resuLts o~ the ele.~.trophor2~ic separations on electrophoretic films~ ~0 and the total isoenzyme an-alyses can be- com~ined to determi~e the ac.tivity re~re-sented by any particular band of patterns having more than one band. Using a spectrophotometer, the optical density o~ each hand in a patter~ can be determined as well as the total optical density for aLl the bands. From this in-formation~ the percentage optical densities of each band can be calculated, w~ich when multiplied by the total isoenzyme activity, gives the activity represented by each li band.
As di~cussed above, the preferred standard and control cells are mouse L ~ells and HeLa cells, respec-tively. Extracts of these cells for incorporation in the kit can be prepared by centrifuging, washing and suspending the cells in a buffer compcsed of snmM Tris, pH 7.5, lmM
EDTA and 0.1% Triton X~10~, disrupting the cells by foxce-full.y ex~ressin~ them through.a pas.teur pipet threc times, centrifuging the crude ex.tract and removing the super-natant li~ui~. ~he supernatant liqui~ is then placed in containers.32 or 34 as appropriate and.freeze-dried in the containers.

Although specific embodiments of the invention have been described and illustrated, it is to be understood that modifications can be made without departing from the invention's spirit a~d scope. Thus, although the invention has been illu~trated with.reference to isoenzyme analysis, it is equally applicable to all types of elec~rophoretic-ally separable substances. Similarlyt although thè se-quencing code has been illustrated using letters of the alphabet a~ the symbols, other symbols can be used~ In the same vein, other grids, data sheets, and reagent coding systems can be sub~tituted for those illus~rated~

Claims (41)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS.

1. An electrophoretic method for identifying cells comprising the steps of:
a) simultaneously performing on an electrophoretic film, multiple electrophoretic separations on a sample from a control, a standard population of cells and at least one sample from an unknown population of cells each of the samples containing at least some electrophoretically separable substances which are characteristic of all the cells;
b) developing the electrophoretic separations of control, standard, and unknown cells with regard to an electrophoretically separable, charcteristic substance;
c) comparing the developed separations for the control and standard samples to determine the range of separation for the characteristic substance under the conditions used; and d) based on the range of separation so determined, comparing the separations for the unknown and standard samples to identify the unknown cells.

2. The method of Claim 1 wherein the electrophoretically separable substances are proteins.

3. The method of Claim 2 wherein the electrophoretically separable substances are isoenzymes.

4. The method of Claim 3 wherein the isoenzymes are purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dahydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

5. The method of Claim 1 wherein the control population of cells is a population of mouse L cells.

6. The method of Claim 5 wherein the standard population of cells is a population of HeLa cells.

7. The electrophoretic method of Claim 1 comprising:
a) performing electrophoretic separations on a series of samples from the unknown population of cells;
b) developing the electrophoretic separations of the unknown samples with regard to a series of different, preselected characteristic substances;
c) determining from the developed samples the eletrophoretic mobility exhibited by the unknown cells to be identified for each of the preselected substances;

d) assigning to the electrophoretic mobility for each substance so determined a symbolic representation;
e) arranging the symbolic representations in a pre-determined order to form a coding sequence for the unknown population of cells; and f) comparing the coding sequence of the unknown population of cells with a compendium of predetermined coding sequences for known cells to identify the unknown cells.

8. The method of Claim 7 wherein the electrophoretic mobility is determined using a grid having a plurality of spaced lines by which the symbolic representations are assigned.

9. The method of Claims 7 or 8 wherein the symbolic representations are letters of the alphabet.

10. The method of Claims 7 or 8 wherein the electro-phoretically separable substances are proteins.

11. The method of Claims 7 or 8 wherein the electro-phoretically separable substances are isoenzymes.

12. The method of Claims 7 or 8 wherein the series of electrophoretically separable substances consists of purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

13. The method of Claim 7 wherein the electro-phoretically separable substances are developed to produce a group of similar colors.

14. The method of Claim 13 wherein the elecirophoretically separable substances are developed to form an insoluble formazan which is colored purple-blue or purple-red.

15. A method for identifying cells comprising the steps of:

a) electrophoretically separating extracts from standard cells, control cells and the unknown cells to be identified simultaneously on separate tracks of an electrophoretic film;
b) developing the electrophoretic film with regard to a first preselected electrophoretically separable characteristic substance using a reagent specific for that substance to produce a pattern of one or more bands of an insoluble product in each track for which the substance is present in the extract, the pattern, where present, being indicative of the extent of electrophoretic migration of the first preselected substance for the standard, control, and unknown cells;
c) interpreting the developed electrophoretic film by reference to grid having a plurality of spaced lines thereon and having a sequence of symbols associated with the lines, the interpretation occurring by aligning the film with the grid by reference to the developed pattern in the track for the standard cells and determining by reference to the grid first and second symbols corresponding to the developed patterns in the tracks for the extracts from the control cells and the unknown cells respectively;
d) recording the first and second symbols so determined at first and second locations, respectively, on a data sheet;
e) repeating steps a, b, c and d for a predetermined number of additional electrophoretically separable characteristic substances, the symbols determined for these substances being recorded on the data sheet at predetermined locations located relative to the first and second locations to form a first coding sequence for the control cells and a second coding sequence for the unknown cells;
f) comparing the coding sequence for the control cells with a reference coding sequence for those cells to determine whether the electrophoretic separations have been performed properly; and g) for properly performed separations, comparing the coding sequence for the unknown cells to be identified with a compendium of predetermined coding sequences for known cells to identify the unknown cells.

16. The method of Claim 15 wherein the electrophoretically separable substances are proteins.

17. The method of Claim 15 wherein the electrophoretically separable substances are isoenzymes.

18. The method of Claim 17 including the additional step of determining the total isoenzyme activity of the cells to be identified for one or more of the isoenzymes for which an electrophoretic separation has been developed.

19. The method of Claim 18 including the additional step of determining the isoenzyme activity represented by each band of a developed pattern having more than one band by apportioning the total isoenzyme activity among the bands based on the relative optical densities of the bands.

20. The method of Claim 15 wherein the electrophoretic films, the reagents and the data sheet are color-coded with respect to the electrophoretically separable substances, one color being used to identify each substance.

21. The method of Claim 15 wherein the symbols are letters of the alphabet.

22. The method of Claims 17, 18 or 19 wherein the isoenzymes are purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

23. The method of Claims 15, 16 or 17 wherein the same insoluble product is produced during development for each of the electrophoretically separable substances.

24. The method of Claims 18 or 19 wherein the same insoluble product is produced during development for each of the electrophoretically separable substances.

25. The method of Claims 15, 16 or 17 wherein the same insoluble product is produced during development for each of the electrophorectically separable substances and wherein the insoluble product is a formazan.

26. A system for identifying cells electrophoretically comprising:
a) extracts from standard and control cells;
b) an extract from unknown cells;
c) a set of reagents for developing characteristic substances in electrophoretic separations of the standard, control and unknown cells each reagent being identified by a code distinct from the codes used for each other reagent, the number of reagents being chosen to permit characterization of the unknown cells;
d) a set of electrophoretic films having means for receiving samples from the extracts of the standard, control, and unknown cells, the films being identified by the codes used for the reagents, one code for each film;
e) an electrophoretic device; and f) means for interpreting the electrophoretic films including means for compiling a sequence of symbols indicative of the separation achieved for the developed characteristic substances of the unknown cells.

27. The system of Claim 26 wherein the symbols are associated with a grid of spaced lines.

28. The system of Claim 26 wherein the compiling means is coded with the codes for the reagents.

29. The system of Claim 26 wherein the reagents develop the electrophoretic separations for the presence of proteins.

30. The system of Claim 26 wherein the reagents develop the electrophoretic separations for the presence of isoenzymes.

31. The system of Claim 30 further including a second set of reagents identified by the same code as the first set of reagents for determining total isoenzyme activity of the cells to be identified.

32. The system of Claims 26, 27 or 28 wherein the reagents are premeasured.

33. The system of Claims 28 or 29 wherein the isoenzymes are purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

34. The system of Claims 26, 27 or 28 wherein the reagents develop the electrophoretic separations to a group of similar colors.

35. The system of Claims 26, 27 or 28 wherein the reagents develop the electrophoretic separations to a group of similar colors, and wherein each of the reagents includes a soluble tetrazolium dye which is reduced to an insoluble formazan which is colored purple-blue or purple-red.

36. The system of Claims 26, 27 or 28 wherein the standard cells are mouse L cells and the control cells are HeLa cells.

37. The system of Claim 26 further including means for decoding the sequence of symbols to determine the identity of the cells to be identified.

38. The system of Claim 37 wherein the decoding means is a compendium of known sequences of symbols for known cells.

39. The system of Claims 26, 28 or 31 wherein the code is a color code.

40. The method of Claims 2 or 3 wherein the control population of cells is a population of mouse L cells.

41. The method of Claims 1, 2 or 3 wherein the control population of cells is a population of mouse L cells and wherein the standard population of cells is a population of HeLa cells.