Concentrations Of Fetal Calf Serum Research Articles

Use of an orthogonal design method to study two-stage chemical carcinogenesis in BALB/3T3 cells.

An orthogonal design method was used to study two-stage chemical carcinogenesis in BALB/3T3 cells. Four factors were studied: different concentrations of the initiator N-methyl-N-nitro-N'-nitrosoguanidine (MNNG); different concentrations of the promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA); different concentrations of fetal calf serum (FCS) and different times at which TPA was added after cell initiation. From the results of three experiments designed by Table L9(3(4)), L8(2(7)) and L16(4(5)), 0.3 microgram/ml MNNG was the highest possible initiating concentration and 0.25 microgram/ml TPA was the minimum effective concentration for promoting activity. There is synergy between MNNG and TPA, the optimum combination in sequential treatment being 0.3 microgram/ml MNNG and 0.25 microgram/ml TPA. The 10% FCS standard concentration was the optimal one; however, below 5% few foci appeared. The time at which TPA was added had little effect on cloning efficiency and transformation frequency. So the use of this orthogonal design in cell culture has many advantages: several factors can be tested simultaneously; it is easy to find the optimal protocol conditions and the dose-response relationship is stable, which enables the reproducibility to be improved. In addition, the different tables proposed may help to reveal unexpected problems.

1,25-Dihydroxyvitamin D 3 inhibitory effect on the growth of two human breast cancer cell lines (MCF-7, BT-20)

1,25 (OH) 2 D 3 has been shown to be able to reduce the growth of several human cell lines. The effect of 1,25 (OH) 2 d 3 on the growth of breast cancer cell lines in relation to their oestrogen (ER) and progesterone (PGR) receptor content has been investigated. The growth inhibition of BT-20 and MCF-7 cell lines is related to the dose of 1,25(OH) 2D 3. It is dependent on the foetal calf serum concentration in the culture medium. At low concentration of 1,25 (OH) 2 D 3 the inhibitory effect is not detectable in the presence of 10% FCS. The rescue of cells from inhibition by serum was less effective when 1,25 (OH) 2 D 3 was present in the medium. The results of [ 3H]thymidine incorporation experiments and DNA measurements are in agreement with the reduction of cell number. Analysis in flow cytometry indicated a reduced number of cells in S phase. These data indicate that 1,25 (OH) 2 d 3 is able to modulate the growth of human breast adenocarcinoma cells regardless of their sex steroid dependency.

Concomitant analysis of osteoblastlike cell migration and proliferation on a serum-enriched growth surface.

Osteoblastlike cell migration and accompanying proliferation on a growth surface precoated with fetal calf serum (FCS) was quantified using a modification of the chemotactic model of Alessandri et al. (1983) and autoradiography. Culture dishes were precoated with 1%, 10%, or 100% FCS and were overlaid with agar. Three-millimeter-diameter wells were cut and first-passage osteoblastlike cells in serum-free medium were seeded into the wells. At 12 and 48 hours, outward migration was quantified by measuring (1) the distance osteoblastlike cells had migrated peripheral to the well margin, and (2) the number of osteoblast-like cells peripheral to the well margin. The data indicated that the migration of osteoblast-like cells was related to time and FCS concentration. More cells migrated a further distance at 48 hours than at 12 hours. In addition, with greater FCS concentrations, osteoblastlike cell migration increased; 3H-thymidine pulse labelling showed no incorporation of label into osteoblastlike cells at 12 hours. However, pulse labelling after 48 hours demonstrated that a small number of nuclei peripheral to the well margin were labelled. The data suggest that proliferation contributes negligibly to the population of osteoblastlike cells peripheral to the well margin. The appearance of osteoblastlike cells peripheral to the well margin is due primarily to migration.

Indirect mechanism of oestradiol stimulation of cell proliferation of human breast cancer cell lines.

The human breast cancer cell line MCF-7 requires oestrogen to produce and promote growth of tumours in athymic mice. In vitro, however, MCF-7 cells proliferate rapidly without supply of oestrogen (Briand & Lykkesfeldt, 1984). Oestrogen stimulation of proliferation of MCF-7 cells can be achieved when the cells are grown at high concentration of newborn calf serum (NCS, 10%) or oestrogen deprived foetal calf serum (10%). The stimulation involves an abolishment of inhibitory activity present in the serum. The oestradiol stimulated cultures grow rapidly for a much longer time period and attain a much higher cell density than the unstimulated cultures. Oestrogen is specific for the promotion of cell proliferation and only oestrogen receptor positive cell lines with a functional oestrogen receptor mechanism can be stimulated. We assume that oestradiol acts directly on the cells and via the oestrogen receptor mechanism induces the synthesis of a substance which abolishes the inhibitory activity in serum. We call this mechanism of action an indirect stimulation of cell proliferation. A similar mechanism may exist in vivo since we find that serum from athymic mice contains a growth inhibitory activity towards MCF-7 cells and the inhibitory effect can be abolished by oestradiol.

A cytotoxic substance (CTS-51) produced by human buffy coat cultures stimulated by staphylococcal enterotoxin B: further characterizations and combined action with interferon.

A recently recognized unique cytotoxic substance, CTS-51, was tested for the hear or acid stability, trypsin digestion and dialysis. Moreover, influences of elevated incubation temperatures or serum concentrations of medium on the cytotoxic activity of CTS-51, and the combination effects of CTS-51 and human leucocyte interferon (HuIFN-alpha (Le)) were investigated. The cytotoxic activity of CTS-51, which is promoted by a small molecule easily passable the dialysis membrane, was found to be very stable to heat (even at 100 degrees C for 30 min) or acid (pH 2.0 for 24 hr at 4 degrees C) treatments. The treatment with 0.75% trypsin for 1 hr did not diminish the CTS-51 activity. The susceptibility of Daudi lymphoma cells to the antiproliferative action of HuIFN-alpha (Le) was further potentiated by treating the cells with CTS-51 for 16 hr. On the other hand, the CTS-51 activity which was revealed to be prescribed by its concentration in the medium, was not potentiated at 39 degrees C when compared to that at 37 degrees C in contrast to HuIFN-alpha (Le) action, and was reduced according to the increase of the fetal calf serum concentration in the medium.

Cell cycle effects of iron depletion on T-47D human breast cancer cells

T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS.

Diclofenac sodium, a negative chemokinetic factor for neutrophil locomotion

Diclofenac sodium, a non steroidal anti-inflammatory agent, was studied for its influence on the locomotion of human polymorphonuclear neutrophils (PMN), in an attempt to define the mechanism governing the drug's anti-inflammatory properties. PMN locomotion was measured by the agarose technique under two conditions of stimulation of cell migration: in the presence of a gradient of stimuli (chemotaxis) and in the presence of various amounts of stimuli incorporated in the gel (chemokinesis). At concentrations below 100 μg/ml, diclofenac in the gel reduced, in a dose-dependent manner, the directed locomotion of PMN induced by a gradient of C 5a-activated serum, peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Klebsiella pneumoniae culture supernatant (KPCS). Diclofenac also inhibited the random locomotion of unstimulated PMN, as well as the PMN chemokinetic activity induced by various amounts of FMLP or activated serum. Inhibition of PMN locomotion by diclofenac decreased when the concentration of the stimulant was raised; this inhibition was inversely related to the concentration of heat-inactivated fetal calf serum in the medium. The directed locomotion and chemokinesis of PMN, induced by FMLP were also reduced in PMN preincubated with diclofenac before migration, suggesting a direct cellular effect of diclofenac. On the other hand, diclofenac did not affect the changes in shape induced in floating PMN by FMLP or activated serum. The observation that diclofenac did not alter the ingestion rate of bacteria by PMN indicates that this drug is not cytotoxic for PMN. Consequently, diclofenac reduces PMN locomotion by interfering with the PMN chemokinetic activity. Diclofenac is an anti-inflammatory drug possessing the original property of acting as a negative chemokinetic agent, for migration of both stimulated and unstimulated PMN. It should therefore be a useful tool for analyzing the elements controlling PMN locomotion speed.

Studies of interferon as a regulator of hematopoietic cell proliferation.

The presence of interferon (IFN) in normal bone marrow and its abnormal production in aplastic anemia suggest that IFN may have normal regulatory roles and implicates them in the pathophysiology of bone marrow failure. We studied the effects of recombinant IFN (r-IFN) on hematopoietic colony formation in methylcellulose cultures of human bone marrow. Both recombinant IFN-gamma (r-IFN-gamma) and recombinant IFN-alpha (r-IFN-alpha) were potent suppressors of myeloid (CFU-C-derived) colony formation, with 50% inhibition occurring at 291 u/ml for r-IFN-gamma and 275 U/ml for r-IFN-alpha. Small amounts of r-IFN-gamma acted synergistically with r-IFN-alpha; as little as 5 U/ml of r-IFN-gamma increased inhibition of CFU-C-derived colony formation by r-IFN-alpha over threefold. Conversely, small amounts of r-IFN-alpha did not affect inhibition by r-IFN-gamma. Inhibition by r-IFN was highly dependent on culture conditions: reduction of the fetal calf serum concentration from 30% to 20%, a change that did not alter the plating efficiency of control cultures, significantly enhanced the action of r-IFN-gamma. Competition between positive hematopoietic factors and r-IFN was further demonstrated as increasing amounts of human placenta-conditioned media, used as a source of colony-stimulating activity, also partially blocked r-IFN inhibition. To determine if r-IFN could directly inhibit the proliferation of a progenitor cell, cells isolated from immature BFU-E-derived colonies, a population enriched for late erythroid progenitors and free of auxiliary cells, were tested; similar inhibition by r-IFN-gamma was observed with these isolated erythroid progenitors as with total bone marrow CFU-E. Although small amounts of r-IFN-gamma also increased inhibition of bone marrow CFU-E-derived colony formation by r-IFN-alpha, no synergy was demonstrable with isolated erythroid progenitor cells. Therefore, even though r-IFN can directly inhibit proliferation of a progenitor cell, auxiliary cells may be required for synergy between r-IFN-gamma and r-IFN-alpha.

Effects of Proliferation on the Decay of Thermotolerance in Chinese Hamster Cells

Development and decay of thermotolerance were observed in Chinese hamster HA-1 cells. The thermotolerance kinetics of exponentially growing and fed plateau-phase cells were compared. Following a 10-min heat exposure at 45 degrees C, cells in both growth states had similar rates of development of tolerance to a subsequent 45-min exposure at 45 degrees C. This thermotolerant state started to decay between 12 and 24 hr after the initial heat exposure. The decay appeared to initiate slightly sooner in the exponentially growing cells when compared to the fed plateau-phase cells. During the decay phase, the rate of thermotolerance decay was similar in the two growth conditions. In other experiments, cells were induced to divide at a slower rate by chronic growth (3 months) in a low concentration of fetal calf serum. Under these low serum conditions cells became more sensitive to heat and the rate of decay of thermotolerance remained the same for exponentially growing cells. Plateau-phase cells were also more sensitive, but thermotolerance decayed more rapidly in these cells. Although dramatic cell cycle perturbations were seen in the exponentially growing cells, these changes appeared not to be related to thermotolerance kinetics.

The differentiation of L5/A10 myoblast cell line (a subclone of L5 line) is controlled by changes of culture conditions

We report here that it is possible to induce differentiation in a subline of L5 myoblast line (L5/A10) by manipulating the culture media. When L5/A10 myoblast are cultured in F 14 supplemented with 10% fetal calf serum the cells grow with a division time of 12 h and reach confluency at a cell density of approximately 2.4 × 10 5 cells per cm 2, without undergoing differentiation, characterized, morphologically, by formation of multinucleated fibers, and biochemically, by the synthesis of muscle specific proteins such as creatine phosphokinase or myokinase. However, cells, grown in F 14 + 10% fetal calf serum, will undergo regular differentiation after a limited number of division when transferred to F 14 medium supplemented with limiting concentrations (1–2%) of fetal calf serum. Investigations of the biochemistry of myoblast differentiation in cell culture will be facilitated by the availability of a cell line that can undergo differentiation under controlled conditions.

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects.

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.

Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line.

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.

Effect of in vivo administration of different adjuvants on the in vitro candidacidal activity of mouse peritoneal cells

The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 °C was called 1 CA 50 unit. CA was expressed in CA 50 units per 10 6 PC. Optimal reduction of the number of viable Candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable Candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1–20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 ( Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldiocta-decylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymor-phonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth.

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.

Clonal growth of human tumor xenografts. First experiences in drug testing.

Human tumors growing as nude mice xenografts were investigated for growth in a clonogenic assay. Colony formation of more than 30 colonies per plate was obtained in 47 of 63 tumors examined (74.6%). The growth rate was enhanced by raising the concentration of fetal calf serum in the culture medium. Plating efficiency ranged from 0.0003% to 5.5%. To study the applicability of this model for screening new anticancer drugs, preliminary studies in drug sensitivity were conducted with continuous incubation of different anticancer drugs in a dose range of 1/10 to 10 times the maximum achievable plasma level. The highest concentration almost always led to a complete suppression of colony growth. In our opinion the procedure described may be a useful combination of two methods in predictive drug testing, provided drug concentration and mode of drug incubation are adequately considered.

Guinea pig endothelial cells in culture.

ABSTRACTIn pursuit of a model for the studies of endothelial cells in a readily available experimental animal, culture methods and the in vitro characteristics of endothelial cells isolated from guinea pig aorta are described. Endothelial cells were harvested by trypsinization with perfusion techniques and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Their passage cultivation was possible for more than six months and they have maintained the in vitro morphological characteristics of endothelial cells.The cell doubling time of the passaged endothelial cells was 115–131 hours. The growth of guinea pig aorta endothelial cells was satisfactory in Eagle's minimal essential medium containing guinea pig serum and fetal calf serum, but not horse and calf serum. Of the various concentrations of fetal calf serum in Eagle's minimal essential medium, 3% fetal calf serum did not activate cell growth and 10% fetal calf serum induced the best growth; in 20% concentration of fetal calf serum the cell growth rate decreased as compared with the rate in 10% fetal calf serum. DNA synthesis (3H‐thymidine uptake) of the cells in 20% fetal calf serum was less than that in 10% fetal calf serum. These results have no relation to cell generation nor to the cell density. With respect to the type of media supplemented with 10% fetal calf serum, Dulbecco's modified Eagle's medium, RPMI −1640 and Eagle's minimal essential medium were satisfactory for the cultivation of guinea pig aorta endothelial cells, but Medium‐199 was unsuitable. By adding endothelial cell growth supplement (75, 150 mcg) or fibroblast growth factor (100, 200 ng) to Eagle's minimal essential medium containing 3% fetal calf serum, the cell growth was stimulated approximately three times. Fibronectin‐coated wells did not activate cell growth.

Effects of Extracellular Calmodulin and Calmodulin Antagonists on B 16 Melanoma Cell Growth

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.

Non-neuronal cells of rat hypothalamus in dissociated cell culture. Evidence that neurophysin modulates growth and DNA synthesis of non-neuronal cells.

The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture. Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum). Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium. DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium. At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin. This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.

Effects of chick brain extract on the proliferation of chick neuroblasts cultured in media supplemented with low and high serum concentrations.

The proliferative activity of chick neuroblasts cultured in a medium containing a low (5%) or a high (20%) concentration of fetal calf serum (FCS) was analyzed and the influence of a chick brain extract was investigated. Morphological observations and tritiated thymidine incorporation measurements have shown that neuroblasts from 6 day-old chick embryo cerebral hemispheres proliferate more actively in the medium with 5% FCS compared to the medium with 20% FCS. The medium containing 5% FCS favoured the maintenance of neuronal cells in a neuroblast stage as shown by electron microscopy. The stimulatory effect of brain extract on the proliferation of neuroblasts is stronger in the low serum culture condition. These findings indicate that a low serum-containing medium is an adequate condition to study neuronal proliferation and effects of growth factors on these cells.