Development of a method for measuring cell number: Application to ...

Cytometry 17:274-276 (1994)

0 1994 Wiley-Liss, Inc.

TECHNICAL NOTE

Development of a Method for Measuring Cell Number: Application to CNS Primary Neuronal Cultures' Cinzia Volonte," Maria Teresa Ciotti, and Luca Battistini Institute of Neurobiology, CNR, Rome, Italy Received for publication January 19, 1994; accepted M a y 11, 1994

In the present work we further develop neuronal cultures and show its direct a method for counting cell number that is comparison with alternative means for totally independent from the permeabil- cellular quantification. The technique is ity or uptake conditions of the cells, from fast, does not require tedious procedures the state of activation of intracellular en- or long washes, and offers advantages zymes, and from cellular metabolism. We such as a high sensitivity and no backprovide a visual characterization of the ground. c 1994 Wiley-Liss, Inc. method and show that it is highly suitable for cells not growing as monolayers as well as for cultures containing numer- Key terms: CNS primary neurons, rat ous aggregates. We also extend the appli- cerebellar granules, cellular quantificacability of this method to CNS primary tion, nuclei

The study of the biological responses to several different stimuli often requires the measurement of parameters such as cellular survival and/or proliferation, therefore establishing the importance of rapid and reliable means for directly or indirectly evaluating cell numbers. Several different techniques have been devised for this specific purpose. Some methods use radioactive substances, requiring exposure, handling, and disposal of radioisotopes (l),and others rely on either intracellular uptake and metabolism of dyes (3,7), or release of protein and enzymes after cell lysis (1, 4). All these methods are not easily applied under experimental conditions that affect cellular permeability or after exposure of the cells to drugs and biological agents that modulate cellular metabolism and enzymatic activities. In general, the applicability of these methods is limited by the small signal-background ratio often provided, or by the difficulties of direct comparisons among different cellular systems or different states of the same cellular type. To overcome the above restrictions, in the present work we have further developed a method that was first adopted for the cellular quantification of tumor cells (8) and is based on the direct count of cellular nuclei obtained after exposure of cells to a detergent-

containing solution dissolving cell membranes and cytoplasm.

MATERIALS AND METHODS Materials included fetal calf serum (GIBCO, Grand Island, NY) and MTT, (3-(4,5-dimethylthiazol-2-y1)2,5-diphenyl tetrazolium bromide (Sigma, St. Louis, MO).

Culture Conditions Neuron-enriched primary cultures from Wistar 8-dold rat cerebellum were prepared a s previously described (5). Cells were seeded (2.5 x lo6 cellsidish) on poly-L-lysine-coated 35-mm plastic dishes. The culture medium was Eagle's basal medium (BME), supplemented with 25 mM KCl, 2 mM glutamine, 0.1 mgiml

'A hhreuiations: LDH, lactic dehydrogenase; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphatebuffered saline. 'Address reprint requests to Cinzia Volonte, Institute of Neurobiology, CNR, 00137 Rome, Italy.

METHOD FOR MEASURING CELL NUMBER

of gentamycin, and 10% heat-inactivated fetal calf serum.

Glutamate Exposure Exposure of cells to different concentrations of glutamate was carried out a s described (2) a t room temperature in 1ml of Locke’s solution (154 mM NaC1,5.6 mM KC1, 3.6 mM NaHCO,, 2.3 mM CaCl,, 5.6 mM glucose, 5 mM Hepes, pH 7.4). After incubating the cells for 30 min, the Locke’s solution was replaced with culture medium lacking serum and the dishes were returned to the incubator until the following day, for the cellular quantification. Cell Counts The culture medium was removed and replaced with 1 ml of a detergent-containing lysing solution (0.5% ethylhexadecyldimethylammonium bromide, 0.28% acetic acid, 0.5% Triton X-100, 3 mM NaC1, 2 mM MgCl,, in PBS pH 7.4 diluted 1110).After -2 min, the cells were collected and the solution consisted of a uniform suspension of single, intact, viable nuclei that were then quantified by counting in a hemacytometer. Broken or damaged nuclei were not included in the counts. In all cases, triplicate plates were scored and counts represent means L SEM (n = 3). LDH Assay LDH released by cells was measured in 100 p1 of culture medium lacking serum. Sample of media were added to 50 mM sodium lactate, 1.4 mM NAD, in 275 mM final NaPO, buffer, pH 7.6, in a final volume of 1 ml. The absorbance of the reaction mixtures was measured at 340 nm, after a n incubation a t 37°C for 30 min. The difference in absorbance between samples originating from cells exposed to 100 and 0 pM glutamate represents 100% of cell death. Measurements were performed in triplicate and data are expressed as % of alive cells and represent means L SEM (n = 3). Colorimetric MTT Assay MTT colorimetric assay was performed using the method of Mosmann (7). The differences in absorbance between samples originating from cells exposed to 0 and 100 pM glutamate represent 100% of viable cell. Measurements were performed in triplicate and data are expressed a s % of alive cells and represent means ? SEM ( n = 3). RESULTS AND DISCUSSION The aim of this work is to further develop the use of a previously adopted method for counting cells (8,9),by providing its visual characterization, by extending its applicability to CNS primary neuronal cultures, and by comparing it to commonly used means for quantification of cell number. Since the method is based on the count of nuclei obtained with the use of a detergent-containing solution dissolving cell membranes and cytoplasm, we first in-

275

vestigated the appearance of the nuclei after having used such a solution. A uniform suspension of single nuclei appearing as phase-bright intact circles surrounded by a dark ring is obtained from healthy cells (Fig. 1).When the cells are dying, either by exposure to a neurotoxic agent or by apoptotic death, the vast majority of the nuclei are dissolved by the detergent-containing solution (data not shown). In contrast to cells stained with propidium iodide (Fig. 1insert) or several other dyes (data not shown), the nuclei obtained with this technique are very easy to quantify even when they are originating from cellular aggregates (Fig. 1). For this reason, we have successfully applied this method to the cellular quantification of primary neuronal cultures originating both from cerebellum and cerebral cortex a t different developmental ages (Fig. 1, Table 1). In order to directly compare this method to previous methods for cellular quantification, as shown in Table 1A, primary cerebellar granule neurons were exposed to increasing concentrations of the neurotoxic amino acid glutamate and cell viability was measured either by direct count of intact nuclei, by activity of various dehydrogenase from active mitochondria (71, or by release of LDH after cell lysis (4). With all three methods tested, a n increase in the concentration of glutamate resulted in a decrease in the neuronal survival, with -40-50% of viable cells obtained at approximately 10 pM glutamate. This value is in agreement with those previously reported (6), and moreover, the three methods elicited comparable quantitative data for neuronal survival (at all the concentrations of glutamate that have been tested). In Table 1B we show the result of a time course experiment. Cerebellar granule neurons were plated a t a density of 2.5 x lo6 cellddish and viable intact nuclei were counted at 2 h and at 1, 3, 6, 9, and 12 d after plating. As previously confirmed also by the propidium iodide staining procedure (5), soon after plating there was a gradual loss of neurons that culminated at day 9 with -20-30% cell death. At days 11-12, there was instead a drastic increase in the cell mortality and only -10% of the cells were surviving in culture. In conclusion, our study shows that the method of counting nuclei is precise, reliable, and yields results that are highly comparable to alternative methods generally adopted for the quantification of cell number. Our data furthermore support a more general applicability of this method to the quantification of cultures whose cell bodies are often not distinguishable from one another and that can differentiate in vitro (under the stimuli of many extracellular factors) or undergo drastic modulations of their physiological parameters (such as membrane permeability or uptake and state of activation of intracellular enzymes). Furthermore, being based on the direct count of cellular nuclei, this method allows a comparison among various cell types even eliciting different metabolic conditions. In addition, the method is fast and does not require tedious

276

VOLONTEETAL

FIG.1. Cellular quantification by count of intact viable nuclei: visual inspection. Neuron-enriched primary cultures from 8-day-old rat cerebellum were plated a t 2.5 x lo6 cellsidish and after 8 d were collected in 1 ml of detergent containing lysing solution. The nuclei

were visualized in a hemacytometer with a phase-contrast light microscope. Untreated cells stained with propidium iodide, which appear as aggregates and are therefore difficult to count (insert),are shown for comparison to the isolated nuclei.

Table 1 Cellular Quantification"

1. El-Battari A, Muller JM, Fantini J, Bellot F, Tirard A, Ducret F,

A. % of alive cellsb JLMglutamate Nuclei MTT 0 100 f 3 100 -c 9 10 38 2 7 51 i 3 25 25 f 4 22 t 6 50 321 9+3 100 3 k 1 0 B. No. of intact nuclei ( X lo4)" 2h 245

k

5

1 DIV 227 2 3

3 DIV 204? 7

6 DIV 201. ? 8

9 DIV 184 2 10

LITERATURE CITED

LDH 100 t 5 41 t 4 20 ? 5 5*5 0

2.

3. 4.

12 DIV 22 2 4

aCerebellar granule neurons were plated at 2.5 x loficells/ dish. In all cases, triplicate plates were scored and values re resent means + SEM (n =3 ). 'At 8 d in vitro (DIV) cells were exposed to different concentrations of glutamate and at 9 DIV were assessed for cell survival by count of nuclei, measurement of released LDH activity and MTT colorimetric assay. 'Intact viable nuclei were counted at different times after plating.

procedures, long washes, expensive compounds or instruments, and it offers advantages such as high sensitivity and practically no background. Finally, the method can constitute also a good means of preparing isolated nuclei for flow cytometry.

ACKNOWLEDGMENTS We thank Dr. D. Mercanti for useful discussions.

5.

6.

7 8.

9

Marvaldi J : Monensin and tunicamycin-induced inhibition of HT 29 cell spreading and growth. J Cell Sci 80:269-280, 1986. Favaron M, Manev H, Alho H, Bertolino M, Ferret B, Guidotti A, Costa E: Gangiosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex. Proc Natl Acad Sci USA. 35:7351-7355, 1988. Hansen MB, Nielsen SE, Berg K: Re-examination and further development of a precise and rapid dye method for measuring cell growth / cell kill. J Immunol Meth 119:203-210, 1989. Koh JY, Choi DW: Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Meth 20:83-90, 1987. Levi G, Aloisi F, Ciotti MT, Thangnipon W, Kingsbury A, Balazs R: Preparation of 98% pure cerebellar granule cell cultures. In: A dissection and Tissue Culture Manual of the Nervous System, Shahar A, deVellis J, Vernadakis A, Haber B ieds).Alan R. LISS, New York, 1989, pp 221-214. Manev H, Favaron M, Siman R, Guidotti A, Costa E: Glutamate neurotoxicity is independent of Calpain I inhibition in primary cultures of cerebellar granule cells. J Neurochem 57:1288-1295, 1991. Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assays. J Immunol Meth 6555-63, 1983. Soto AM, Sonnenschein C: The role of estrogen on the proliferation of human breast tumor cells (MCF-7). J Steroid Biochem 23(1):87-94, 1985. Rukenstein A,, Rydel, RE, Greene LA: Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-indenpendent mechanisms. J Neurosci 11:2552-2563, 1991.