Simplified Culture and Chromosome Preparations of Primate Leukocytes

Plasma recovered from 1 ml of primate peripheral blood by centrifugation is planted in a medium consisting of 80% TC-199 and 20% fetal bovine serum, to which 0.125 ml of phytohaemagglutinin/5 ml is added. The pH is adjusted to 7 with 10% NaHCO₂. The mixture is incubated 68 hr, Colcemide to give 1 μg/ml is added, and incubation continued for 4 hr. Following centrifugal separation, the cells are given a hypotonic treatment with 0.75% sodium citrate for 15 min, then centrifuged again and fixed in 3:1 methanol-glacial acetic acid, 3 changes. Tiny drops of the cell suspension are placed on a slide, spread by blowing, and air dried. The preparations are stained with 15% Giemsa solution in methyl alcohol. The method has been successfully used in 256 specimens from 25 different species.     

Leukocytes Cultured from Small Inocula of Whole Blood and the Preparation of Metaphase Chromosomes by Treatment with Hypotonic KCL

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO₃, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying method, and staining with 1% natural orcein (G. T. Gurr) in 60% acetic acid. Dehydration and covering completed the preparation. KCI, 0.075 M, has been used advantageously in the above way and for cells cultured by other means from skin and other organs of man and other mammals. Combined advantages of the method are: culture of leukocytes from small volumes of whole blood, with very few failures to obtain mitotic cells; a medium which can be stored frozen in culture vials, and which in a simpler form is usable for long term culture of other cell types; and the use of KCI for hypotonic treatment.     

Volatile Flavor Components of ZINGIBERIS RHIZOMA (Zingiber officinale Roscoe)

The toxicity of dimethyl sulfoxide (Me₂SO) was examined in HeLa cells cultured at 37°C for up to 72 hr. The growth of the cells was measured by a colorimetric method with the use of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which gave good correlation between the cell number and the color development from the reduction of MTT under suitable conditions. When the initial number of cells was 3 × 10⁴/ml, Me₂SO at 1% or less had no apparent effect on prolifiration for up to 48 hr of incubation, but in longer incubations, cell growth was repressed. When the initial number of cells was 3 × 10⁵/ml, the effect of Me₂SO was similar.     

Isolation of alveolar type II cells by centrifugal elutriation.

Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9 +/- 1.0 x 10(6) cells per rat lung (mean +/- S.D., n=30) were recovered of which 86 +/- 6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101 +/- 21 nmol per hr . 10(6) cells (mean +/- S.D., n=4), and their oxygen consumption increased only 10% after 10 mM sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugral elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells.     

Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Leukocyte Culture and Chromosome Preparations from Adult Dog Blood

Plasma is obtained from dog blood after 3 hr settling in a syringe. Portions of the plasma (0.5-1.0 ml) are added to 4 ml of a medium consisting of 17 parts of BME Spinner, 3 parts of calf serum, 0.5 parts of glutamine, 0.5 parts of penicillin-streptomycin, and 0.1-1.0 parts of Scarlet Runner bean phytohemagglutinin. Colchicine, 0.1 ml of 10:1 stock solution, is added after 72 hr and incubation continued for 2 hr, then centrifuged 5 min at 700 rev/min. The supernatant is discarded, 3 ml of distilled water added, and the cell suspension centrifuged again. The supernatant is discarded and the fixative, consisting of 45% glacial acetic acid allowed to act for 0.5 hr. Acetic-orcein stains of smears were very satisfactory.     

Chromosome preparations in the field from mammals long after death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01% colchicine solution to 5 ml of medium-cell suspension. After 1/2-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KCl. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanol:acetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Giemsa, if required after pretreatment of the preparations for banding, e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Preparation of Poliovirus Labeled with Phosphorus-33

Phosphorus-33 ((33)P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 muCi of (33)P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10(10) to 5 x 10(10) plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 x 10(-4) to 10(-3) disintegrations per min per PFU have been obtained from 1.5 x 10(8) to 3.0 x 10(8) HeLa cells.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.     

A stable propidium iodide staining procedure for flow cytometry

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.     

An Air-Drying Procedure for Mammalian Male Meiotic Chromosomes, Following Softening in Gluconic Acid and Cell Separation by An Ethanol-Acetic Mixture

A I cm³ sample of tubules from testes is placed in 5 ml of 0.7% Na-citrate for 20-30 min, then 5 ml of glacial acetic acid is added, mixed well, and allowed to stand for 30 min. The mixture is centrifuged, the supernatant removed, and 3 ml of 3 M gluconic acid is mixed with the tissue and allowed to act for 3 hr. The gluconic acid is removed with a pipette and the tissue is suspended in 5 ml of a freshly made 1:1 absolute ethanol-glacial acetic acid mixture. The tissue is drawn into and discharged from a syringe several times through an 18, a 20, and finally a 22 gauge needle to separate and suspend the cells. The cells are centrifuged and resuspended several times in fresh fixative to remove the gluconic acid. Finally, the cells are suspended in sufficient fixative to give a smear of suitable density, and air-dried preparations are made, or the suspension may be stored at 0-5 C for several days. The cells can be stained by any of the usual stains for chromosomes. This technique results in the improved spreading produced by the air-drying technique and permits recovery of all stages of meiosis and mitosis present.     

The influence of dehydroepiandrosterone on basal and gonadotrophin-stimulated testosterone secretion by Mongolian gerbil interstitial cells incubated or superfused in vitro

1. Testosterone secretion by Mongolian gerbil interstitial cells incubated in the absence of HCG linearly increased with cell concentration (1 x 10(5) cells: 0.6 ng/4 hr, 10 x 10(5) cells: 8.0 ng/4 hr). Addition of 100 mIU HCG resulted in a drastic increase of testosterone secretion which was linear between concentrations of 1 x 10(5) and 4 x 10(5) cells. 2. Compared to HCG-stimulated testosterone release, secretion was significantly higher by cells incubated with 60-100 ng DHEA. 3. During the 4-hr incubation period, 53-69% of added progesterone and 72-88% of added dehydroepiandrosterone (DHEA) were converted to testosterone by cells freshly prepared or stored for 1-3 days at 4 degrees C. On the other hand, prolonged storage at 4 degrees C resulted in a marked decrease of HCG-stimulated testosterone secretion. 4. Testosterone secretion by interstitial cells superfused in vitro increased with the length of HCG (100 mIU/ml) application from 0.08 to 0.22 ng/10(6) cells/min (10 and 60 min, respectively). A much faster and pronounced elevation was found when cells were stimulated with DHEA (200 ng/ml: 0.06-0.80 ng/10(6) cells/min, 0 and 20 min, respectively). 5. After interstitial cells have been stimulated with a DHEA (200 ng/ml) pulse for 30 min and then superfused with medium only for an additional 30 min, testosterone secretion remained significantly elevated and could not be further stimulated by superfusing medium which contained as much as 100 mIU/ml HCG.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific partial depletion of graft-vs-host activity by incubation and centrifugation of mouse spleen cells on allogeneic spleen cell monolayers.

Spleen cells (from BALB/c mice immunized with the C57BL/6 lymphoma EL4, or from non-immune BALB/c) were incubated on monolayers of [C57BL/6 times BALB/cF1 (B6CF1) spleen cells on polylysine-coated polystyrene Petri plate, for 1/2 hr or for 1 hr at 37 degrees C followed by centrifugation of the monolayers for 5 min at 70 times G to 110 times G at 34 to 37 degrees C. Control monolayers were BALB/c spleen cells. As measured by the Simonsen spleen weight assay in neonatal mice, graft-vs-host (GVH) activity was partially depleted in cell populations nonadherent to B6CF1 monolayers. Residual GVH activity of these nonadherent cells was about half that of cells incubated on the control syngeneic monolayers (the mean of eight experiments was 49% +/- 11% S.D.). Two or three consecutive cycles of incubation and centrifugation did not significantly diminish the residual GVH activity, suggesting that spleen cells with GVH activity are heterogeneous with respect to binding to allogeneic target cells under the above conditions. Cell populations nonadherent to third-part [A times AL]F1 monolayers retained full activity, and cell populations partially depleted of GVH activity in B6CF1 neonates had full activity in third-party [BALB/c times AL]F1 neonates.     

Inhibition of HeLa-S3 cell proliferation and biosynthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)

Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.     

A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment

Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm³ pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture. This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington, D. C. (R810146).     

Culture and Slide Preparation of Leukocytes from Blood of IMACACA

Leukocyte-rich plasma is recovered from monkey blood by centrifugation. Portions of the plasma are planted in a medium consisting of 70% NCTC 109, 10% F 10, 15% fetal bovine serum, 5% human cord serum, and phytohemagglutinin (0.1 ml/5.0 ml of medium). Incubation is 48-72 hr, followed by colchicine 24 hr, hypotonic treatment with water 1-1 1/4 hr, and fixation in absolute methanol-glacial acetic acid (3:1). Permanent spreads are made by ejecting drops of suspension onto iced slides and flame drying. Staining is done in cresyl violet acetate. Because of some inconsistencies encountered in the spreading of monkey cells, alternate methods have been suggested for trial until one is found that works uniformly for the technician.     

Basal and HCG-stimulated testosterone production by interstitial cells of the Mongolian gerbil (Meriones unguiculatus)--I. Influence of preincubation length, medium composition and temperature

In the absence of HCG, production of testosterone by whole testes superfused in vitro was quite constant during the 5-hr superfusion period. Addition of 23-184 mIU/ml HCG caused a significant increase of testosterone production which was apparent from 30 min after start of superfusion. Basal and HCG-stimulated testosterone production by whole testes was significantly higher (400, 1950 ng/testis/5 hr, without and with 100 mIU HCG) than by isolated cells (200, 1350 ng/testis/5 hr). Incubation of isolated interstitial cells in medium 199 supplemented with fetal calf serum (FCS), (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid, HEPES) and 3-isobutyl-methylxanthine (MIX), and in medium 199 without FCS, HEPES or MIX, gave similar testosterone responses. While centrifugation at 8000 g for 2 min drastically diminished testosterone formation by isolated interstitial cells, production was similar by cells incubated in either 0.5, 1.0 or 1.5 ml medium. A significant decrease of testosterone synthesis by isolated interstitial cells was found when cells were stored at 4 degrees C for 2 days and then were incubated at 35 degrees C for 6 hr without or with 1-1000 microIU HCG. While isolated interstitial cells incubated at 5 degrees C did not produce testosterone at all, testosterone production increased to 49.5 +/- 3.9 ng/10(5) cells (30 degrees C) and 24.1 +/- 1.1 ng/10(5) cells (40 degrees C), respectively. HCG-stimulated testosterone production was maximal when interstitial cells were incubated at 34 degrees C.