A METHOD FOR VIABLE CELL COUNT

Submitted by

SUSAN TOLNAI Department of Histology and Embryology Faculty of Medicine University of Ottawa Ottawa, Ontario Canada I. INTRODUCTION Several methods are available to differentiate between live and dead cells in suspensions. The more elaborate procedures include the measurement of endogenous respiration (1), reduction of 2,3,5-triphenyltetrazolium chloride (2) and uptake of tritiated thymidine (3,4). The most widely used procedure is the dye exclusion test. This method is based on the assumption that certain dyes, e.g. trypan blue, methylene blue, acridine orange, eosin, nigrosin, safranin, etc. stain only dead cells while live cells remain unstained ("exclude" the dye) when exposed to these substances in suspensions. Each of the above methods utilize different parameters as indicators of cell viability and therefore results obtained with the different procedures should be carefully evaluated before direct comparisons or correlations are attempted. Among the different vital and supravital dyes the two most frequently employed for viability tests are trypan blue and eosin, described originally by Sampson (5) and Schrek (6), respectively. Although the method using eosin exclusion obtained early and wide acceptance (7), more recently several authors warned against possible pitfalls. Hauschka et al. called attention to the fact that after prolonged storage at low temperatures, many non-viable cells remained unstained in eosin (1), while Eaton et al. observed a decline in the transplantability of tumor cell suspensions before the inability to exclude the dye became apparent (8). The possibility that trypan blue staining indicates only late stages of cell injury also has to be considered. King et al. (9) noted that the loss of capacity for cell division and respiration probably precedes stainability and the functional loss is not revealed by viability tests using trypan blue. However, the careful studies of Allison and Young (10) indicate that diffuse cytoplasmic staining by trypan blue signifies cell death and is preceded by staining confined to phagosomes, discernible in the light microscope as granular staining in the cytoplasm.

E oz ~-O

.E <_|

N m ~'ee
.~_ ~

II.

MATERIALS
Haemocytometer (Neubauer-Spencer Bright Line) 1 White blood cell counting pipette Trypan blue stock solution: 1% trypan blue (vital stain, C.I. 23850) in Hanks' balanced salt solution Hanks balanced salt solution Clinical centrifuge (Model CL) ~ Conical centrifuge tubes

III. PROCEDURE
A. B. C. Add 0.1 ml of Trypan blue stock solution to 0.9 ml of cell suspension. Allow to stand for 10 minutes at room temperature. Centrifuge the cell suspension (5 minutes at 1000 rpm.), remove the supernatant and re-suspend the cells in 9 volumes of fresh Hanks' balanced salt solution. Count the stained and unstained cells in both chambers of the haemocytometer. Minimum of 200 cells should be counted for each sample. Each chamber is divided into 9 large squares by triple white lines. Count the cells in the large centre square and in the four

LU I~m

D.
w z O<{ I,----1 "o

L/ 2_/ ~-

A m e r i c a n Optical Co., R o c h e s t e r , N e w Y o r k I n t e r n a t i o n a l E q u i p m e n t Co., N e e d h a m Heights, Massachusetts

37

corner squares of both chambers; the total number of cells in 10 squares = number of cells in l m m 3. If the cell suspension has to be diluted for convenient counting, use a white blood cell counting pipette. Fill the pipette to the 0.5 mark with the cell suspension and to the 11 mark with the diluting fluid (Hands BSS). Mix well the contents of the pipette, discard the first two or three drops and fill the counting chambers from the cell suspension contained within the bulb of the pipette. To obtain the number of cells in 1 mm 3 multiply the total number of cells counted in 10 large squares by 20.
Number of stained cells x 100 of cells counted

(2) Hoskins, J.M., G.G. Meynell and F.K. Sanders. 1956. A comparison of methods for estimating the viable count of a suspension of tumor cells. Exp. Cell Res. 11:297-305. (3) Boeryd, B., O. Eriksson, F. Knutson, P.M. Lundin and K. Norrby. 1965. On the viability of tumor cells in artificially produced suspensions. Acta path. et microbiol. Scand. 65:514-520. (4) Wojciech, S., J. Kieler and P. Briand. 1967. Vital staining with neutral red and trypan blue of 3 H-thymidine labelled cells prior to autoradiography. Stain Technol. 42:143-146. (5) Sampson, J.J. 1924. Determination of the resistance of leukocytes. Arch. Int. Med. 34:490-502. (6) Schrek, R. 1936. A method for counting the viable cells in normal and in malignant celt suspensions. Amer. J. Cancer. 28:389-392. (7) Hanks, J.H. Wallace. 1958. Determination of cell viability. Proc. Soc. Exp. Biol. Med. 98:188-192. (8) Eaton, M.D., A.R. Scala and M. JeweU. 1959. Methods for measuring viability of ascites cells. Cancer Res. 19:945-953. (9) King, D.W., S.R. Paulson, N.L. Puckett and A.T. Krebs. 1959. Cell death IV. The effect of injury on the entrance of vital dye in Ehrlich tumor cells. Am. J. Pathol. 35:1067-79.

Total number

= % of stained cells in t h e s a m p l e .

IV. DISCUSSION
The advantage of the dye exclusion method for the determination of cell viability is the fact that it is easy to perform and several samples can be processed in a short time allowing adjustments of cell concentrations to be made prior to plating, culture seeding, etc. However, care should be exercised to maintain exact conditions of sample preparation in order to obtain reproducible results. It is also important to note, that trypan blue can bind to serum proteins in the physiological pH range; therefore if the serum concentration in the sample cell suspension is high, it may be necessary to raise the effective concentration of the trypan blue solution (11).

E oz

.~__~|
-Q<~N
~ if) e. I"d

cD

.~r--

V.

REFERENCES

(1) Hauschka, T.S., J.T. Mitchell and D.J. Niedergruem. 1959. A reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at _78~ Cancer Res. 19:643-653.

(10) Allison, A.C. and M.R. Young. 1964. Uptake of dyes and drugs by living cells in culture. Life Sci. 3:1407-1414. (11) Pappenheimer, A.M. 1917. Experimental studies upon lymphocytes. I. The reactions of lymphocytes under various experimental conditions. J. Exp. Med. 25:633-650.
ul I-" ,~r,"

mz
I,----

38