Ml Of Cell Suspension Research Articles

Simple and small-scale breakdown of yeast

A simple and small-scale method for the preparation of homogenate from yeast was developed. The principle of this new method involves the shaking of a small amount of yeast-cell suspension with glass beads on a tower-shaped mixer. When this method is used the cells in 1 ml of cell suspension are broken down and 14 samples can be simultaneously processed under controlled conditions. The degree of cell breakdown, the amount of soluble protein liberated from the cells, and the increase in each enzyme activity in prepared homogenate correlated to the shaking time: maximum values were obtained at 20 min. Enzyme activities in the homogenate were equivalent to or higher than that procured with former methods. This new method is applicable to various species of yeasts.

Variation in Adenosine Triphosphatase Activities in Serial Preparations of Human Erythrocyte Membranes

Erythrocytes provide material that is readily available from human subjects and from which membrane preparations can be made for biochemical studies. Such preparations have been used for the study of various clinical and metabolic conditions. To evaluate such studies it is important to know whether a single membrane preparation provides sufficiently reliable information to judge possible changes in serial samples from the same subject. Our study describes the estimation of three ATPaseS activities in erythrocyte-membrane preparations and their variation in membranes prepared from three subjects over a period of 5 weeks. Human erythrocyte membranes were prepared by the method of Dick et al. (1969) from heparinized 10-20ml samples of blood collected by venepuncture. After centrifugation at l000g for 4min, the plasma was discarded and the cell pellet then washed three times in ice-cold iso-osmotic 156rn~-NaCl/Srn~-buffered Tris, pH 7.4. Washing was by repeated centrifugation at l000g for 4min at room temperature. After one or two washes the buffy coat was removed. The washed cell pellet was resuspended in the cell-wash solution to an approximate 40% haematocrit. A portion of the cell suspension was taken for accurate determination of the haematocrit and the known volume of the remainder haemolysed by addition of I0m-Tris buffer, pH7.4 (34ml of buffer: 6ml of cell suspension) and vortex-mixed briefly. The haemolysate was left at 0°C for 10-15min and centrifuged at 28000g for lO5min in a Beckman L2-65B ultracentrifuge with a swing-out rotor. To obtain reproducibly active preparations, it was necessary for all the post-lysis centrifugations to be carried out between 9 and 10°C. Centrifugation at 28000g for lOSmin produced a red membrane pellet. The membranes were then washed three times by resuspension in IOmM-Tris buffer and centrifuged at 28000g for 1 h. This produced a layer of fluffy, white or faintly pinkish membranes, together with a pellet of cell debris and haemoglobin. The membranes were dispersed by brief vortex-mixing and decanted into a fresh tube for storage, whereas the pellet of debris remaining in the centrifuge tube was washed once in IOmM-Tris buffer by vortexmixing and the wash added to the membranes. ATPase activities were measured by the liberation of PI from disodium ATP (Boeh

Toxicity of Na 251CrO 4 when used to label rat lymphocytes

Lymphocytes preincubated with various activities of Na 2 51CrO 4 were tested for their ability to induce a local, popliteal lymph node graft-versus-host (GVH) reaction. Incubation with 100 μCi of the isotope per ml of cell suspension impaired the GVH activity, 25 μCi gave a reduced response in 1 out of 3 experiments, whereas 10 μCi had no effect. The reduced GVH response was due to radiation rather than chemical damage of the cells. Preincubation of the cells with either cold Na 2CrO 4 or outdated Na 2 51CrO 4 with concentrations of Cr identical to the ones used to label cells with 100 μCi/ml cells did not impair the GVH reactivity. The cellular uptake of Na 2 51CrO 4 showed first-order kinetic over wide concentrations ranges. Mitomycin C-treated lymphocytes showed a more heavily depressed GVH reactivity than would be expected from their moderately reduced ability to accumulate in the lymph nodes. It is recommended that doses of Na 2 51CrO 4 in excess of 10 μCi/ml cells should not be used for functional studies of lymphocytes.

Endotoxin influence on lipolysis in isolated human and primate adipocytes

The influence of E. coli endotoxin on basal and norepinephrine-stimulated lipolysis as well as on cyclic AMP accumulation was studied in human and rhesus monkey adipocytes. The effect of indomethacin on lipolysis was also investigated. Human and rhesus adipocytes exhibited qualitatively similar responses to norepinephrine (0.59–11.8 μ M). There was a positive correlation between cell volume and basal lipolysis in human, but not in rhesus, cells. A positive correlation was also noted between cell volume and the response to endotoxin and to norepinephrine in human cells. The increment in response to norepinephrine after endotoxin treatment, however, was negatively correlated to basal lipolysis. Endotoxin (0.32 μg/0.5 ml of cell suspension) increased basal free fatty acid release and significantly enhanced norepinephrine-stimulated glycerol release from rhesus adipocytes. In human fat cells, basal release of glycerol was stimulated by endotoxin at all concentrations used, whereas basal FFA release was increased at endotoxin concentrations of 0.032 and 0.320 μg/0.5 ml of cell suspension. Endotoxin significantly depressed norepinephrine-stimulated release of FFA at all endotoxin concentrations without significantly influencing concurrent glycerol release. Treatment of rhesus fat cells with endotoxin and endotoxin plus indomethacin resulted in significant increases in cAMP content. In human fat cells, endotoxin alone did not influence cAMP concentration; however, exposure to norepinephrine or to endotoxin plus indomethacin resulted in significantly increased levels of cAMP.

Chapter 1 Preparation of Large Quantities of Pure Bovine Lymphocytes and a Monolayer Technique for Lymphocyte Cultivation

Publisher Summary This chapter discusses the preparation of large quantities of pure bovine lymphocytes and a monolayer technique for lymphocyte cultivation. The lymphocyte preparation begins by removing the tissue surrounding the lymph node with a pair of surgical scissors. The lymph nodes are then inspected macroscopically for the presence of inflammation foci which appear as livid areas of a hard consistency as compared to the brown-yellow color and soft consistency of less reactive lymph nodes. The lymph node is then fixed with two needles at both of the small ends to a stanniol covered cork plate and swabbed once with 70% ethanol. Under sterile conditions the connective tissue capsule is opened with one lengthwise cut on the convex side of the lymph node. Lymphocyte purification is carried out at 37°C. The saline solution in the column is replaced by 200 ml of Eagle's medium plus 10% inactivated calf serum and then 200 ml of cell suspension is added. The cells are allowed to interact with the glass fibers for 30 minutes, after which the cell suspension is slowly removed (3 minutes) from the column.

A packed cell volume (“cytocrit”) method for measuring swelling of rat brain cells exposed to sera of rabbits with allergic encephalomyelitis

A cytocrit method for determining the swelling effect of cytotoxic antibodies was adapted for use with fresh rat brain cells in saline acted upon by sera of rabbits showing paralysis of AE. Detailed descriptions of the procedure, and advantages and disadvantages of the method are presented. Brain cells in balanced saline show swelling; this swelling is reduced by about 76% by adding 0.1 ml of normal serum to each ml of cell suspension. The difference between swelling with normal serum and with AE serum is believed to represent the swelling effect of cytotoxic antibodies in AE serum. Swelling of rat brain cells with AE sera is temperature-dependent. The curve of swelling at 37°C is logarithmic through 210 minutes of incubation. Control swelling is of slightly less magnitude if control sera are used within 10 days of drawing than if used 15–41 days after drawing. Using the same experimental and control sera at 37°C in 3 independent runs under the same conditions, results with control serum agreed within 0.12%; with experimental serum, within 23.8%.

Method for creating rapid cellular temperature perturbation

A temperature perturbation in biological systems has been performed in a simple apparatus. A 5° jump in 0.5 to 0.7 ml of cell suspension was obtained with a 200 joule xenon flash within 400 msec. The jump was “chemically pure” and little or no damage of cells was detected microscopically. The response of cellular NADH to the temperature jump was an initial fast decrease in its fluorescence efficiency, followed by a delayed NADH oxidation with a half-time of several seconds. No more rapid biological relaxation was observed under the conditions studied. The advantages of using a flash heating and of a current pulse heating are discussed.

微生物による海水中の溶存含窒素化合物の微量定量法-I

On the microquantitative determination of ammonia present in sea water, the study was carried out, the marine nitrite former A 6 being used. The results obtained are as follows: 1. Five mililiters of the sample in which particulate matters such as bacteria have been eliminated by means of centrifugation and 0.5ml of cell suspension (0. D.=0.5) of the bacterium A 6 are mixed in the test tube. The mixture being incubated for 2 hrs at 30°C, the cells in it are eliminated by means of centrifugation and the nitrite concentration in the supernatant fluid is measured according to the ordinary method using Griess and Romjns reagent. 2. Ammonia present in sea water can be determined by this method, even in low concentrations such as 0.1-10 N μg at. /1.

The nucleic acid from avian myeloblastosis virus compared with the RNA from the Bryan strain of Rous sarcoma virus.

This paper describes properties of the nucleic acid isolated from the avian myeloblastosis virus (AMV) and compares this nucleic acid with the RNA from the Bryan strain of Rous sarcoma virus (RSV) and its helper virus, Rous associated virus (RAV). The Bryan strain of RSV is defective' and infected cells do not produce mature RSV unless simultaneously infected with an avian leukosis virus such as Rous associated virus (RAV) which functions as helper virus and presumably provides a protein coat for RSV.2 For this reason all preparations of RSV contain a helper virus which is usually present in 4-10 times higher concentrationl 3' 4 of infectious particles than RSV and which has coat properties indistinguishable from those of RSV. Because of the similar properties, RSV has not been separated from its helper virus.4 A recent report from this laboratory5 describes the purification of the mixture designated RSV + RAV and the isolation and preliminary characterization of the viral RNA. Avian myeloblastosis virus, like RAV, is an avian leukosis virus and the two are closely related biologically. Both will function as helper viruses for RSV,2 6 interfere with RSV,4 and produce tumors in chickens. They differ in specific antigens, and only AMV produces acute myeloblastic leukemia.7 The intact virus particles have been shown to have similar morphology, chemical composition, and physical properties.7' 8 Because of the close relation, it might be expected that the two would have similar nucleic acids. However, previous reports9' 10 describe only lowmolecular-weight RNA isolated from AMV, and each of several reports-3 reveals a different RNA base composition. In the present study we find the nucleic acids of AMV and RSV + RAV to be single-stranded RNA, indistinguishable in sedimentation behavior and change in conformation with change in salt concentration and very similar in base composition. Methods and Materials.--Viruses: An AMY strain, kindly supplied by Dr. P. K. Vogt, belonging to the BAI strain A and to the B classification of Vogt,7 and RSV + RAV-86 were used. Production of AMV: Chick embryos on the 12th day of incubation were inoculated intravenously'4 with 104 converting units5 of AMY and allowed to hatch. The chicks that showed a very high leukemic cell count (5 X 105 or more per mm3) in the peripheral blood 10 days after hatching were exsanguinated by cardiac puncture. The blood was collected into hepariin and centrifuged at low speed to pack the cells. The leukemic myeloblasts forming the upper two thirds of the packed cell layer were distinctly separated from the lower layer of erythrocytes. The leukemic cells were isolated and resuspended at a concentration of 1-5 X 106 cells per ml in nutrient medium.5 Ten ml of cell suspension in a 100-mm plastic tissue culture dish was incubated at 40?C in a humidified incubator flushed with a C02-air mixture to maintain the culture pH around 7.3. The cells did not attach to the floor of the plate. At approximately 12-hr intervals for 48-96 hr the cells were removed from the medium by low-speed centrifugation and resuspended in fresh medium. The medium recovered after each period of incubation was centrifuged a second time at 2,000 X g for 15 min to remove cell debris and was then frozen at 80?C until used for virus purification at a later time. Such medium contained about 105 converting units per ml.

The effect of fatty acids on the respiration of Leptospira icterohemorrhagiae.

Measurements of oxygen uptake were done in the Warburg apparatus by conventional methods (Umbreit, Burris, and Stauffer, 1945), at 37 C, in an atmosphere of air. Flasks of 20-ml capacity contained 2 ml of cell suspension and 0.8 ml of substrate; the center wells contained 0.12 ml of 10% KOH. The values shown for oxygen uptake in the curves are cumulative throughout the experimental period. Cell suspensions were prepared from cultures of Lept. icterohemorrhagiae (Wijnberg strain) which had been grown in a slightly modified Stuart's