Tissue culture media

Figure 6, Polarized epithelial cells in culture. Epithelial cells in culture possess an apical surface with microvilli that faces the tissue culture medium (equivalent to the lumenal side of the cells in vivo), and a basolateral surface that faces the tissue culture dish (equivalent to the blood side of the cells in vivo).

Keeping lycopene soluble and unoxidized in the warm, aqueous tissue culture medium over the incubation periods of 12-72 h is problematic. Furthermore, depending on the lycopene preparation, variable amounts remain on the filters used to prevent microbial contamination and hence the... 

A complete assay requires the test material to be investigated at a minimum of three doses together with a positive (untreated) and solvent-only control can be omitted if tissue culture medium is used as a solvent. When two fixation times are used in repeat tests, the positive control is necessary at only one time but the negative or solvent control is necessary at both times. 

Rreisberg, J.I., Pitts, A.M. and Pretlow, T.G. (1977). Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of Ficoll in tissue culture medium. Am. J. Pathol. 86 591-601. 

If the cells are tissne-cnlture cells intended for cell sorting, use tissue-culture medium in which the cells have been growing as the wash solution. The tissue-culture supernatant is withdrawn from the tissue-cnlture flask and filtered through a 0.22-p filter to ensure sterility. The addition of this medium helps cells recover after sorting and increases the growth of cells when they are placed back into tissue culture. 

K-Assay-H202 Assay CL determination, concentration range from 2.44 to 2500 p-gL in buffer and from 39.06 to 10.000 p-gL in tissue culture medium . d... 

In a setup of cell culture, we have found that tissue culture medium salts (Earls salts) can be replaced by NaOH or HCl and water within a range of pH 2-11.3 without changing the 350 mOsmol/kg osmolarity of the medium. Therefore, the experimental setup does not interfere by means of semipermeable membranes with the water content of the cells. 

We have exposed non-confluent ceU cultures with 50,000 cells/mL attached to the cell culture bottle and introduced a medium exchange for 1 h. The medium was exchanged with pH from 2, 3, 4, 5 and 9, 10, 11, 11.3. We have observed acute changes and after a period of 1 h, the pH-conditioned medium was withdrawn and replaced by a tissue culture medium. The overall conclusion of these experiments show that pH must be returned to less than 9 and higher than 5 within less than 60 min and preferably within 30 min. This is achieved by the results are given in Fig. 6.14. 

The use of enzymes, such as papain, to cleave beadxell rosettes has also been investigated chymopapain, used at 200 U/mL of cells (107/mL m TC199 tissue-culture medium) for 10 mm at 37°C, has been found to be most effective and least toxic to KGla human leukemia cells, giving 100% recovery of viable cells (45)... 

An immunogen induces antibodies from many B cell clones, producing a polyclonal antibody response. In contrast, the propagation of an isolated B cell clone produces an antibody of single specificity. However, the problem is that in tissue culture medium, B cells die within a few days of their isolation from, for example, a mouse spleen. To circumvent this problem, immortality can be conferred on B cells by means of viral transformation Epstein-Barr virus can be used. Alternatively, fusion to cancerous cells is carried out to generate hybrids or hybridomas. Generally, the former procedure is used to immortalize peripheral blood B cells and produce human monoclonal antibodies, while myeloma cells are used to produce murine monoclonal antibodies. 

Isolation and Quantitation of Glycosphinqolipids from Cultured Fibroblasts, Plasma Lipoproteins and Tissue Culture Medium... 

In a separate 96- or 384-well plate, prepare serial dilutions of the vims in tissue-culture medium. We have typically used 10-fold serial dilutions, although threefold dilutions may enable a more precise determination of optimal vims concentration. 

Aspirate medium and replace it with 30 pL of tissue-culture medium. 

Replace tissue culture medium by aspirating old medium and adding new 32-37°C medium (15-20 ml T75, 25-35 ml T150) every 2-3 days until cells reach 80% confluence. 

Sutherland GR Fragile sites on human chromosomes demonstration of their dependence on the type tissue culture medium. Science 197 265-266,1977. 

The testes of each rat were decapsulated and gently washed in tissue culture medium (TCM) 199 (Sigma biochemicals M 2154) previously gassed with car-bogen (95 % O2, 5 % CO2). Each decapsulated testis... 

Darby, C. R., Hamano, K., and Wood, K. J. (1993). Purification of monoclonal antibodies from tissue culture medium depleted of IgG.. Immunol. Methods 159, 125-129. 

Male rats used for the perfusion experiments were maintained under established conditions and were fasted 16-24 hours prior to surgery as previously described (35). In the first series of studies the luminal perfusate was at pH 4.2. This perfusate consisted of M199 tissue culture medium which contained a variety of amino acids, vitamins and minerals plus glucose. The perfusate was supplemented (at 110 umolar) with L-histidine HCl, L-cysteine, L-methionine, L-tryptophan, 2-picolinic acid, citric acid, or reduced glutathione. The mixture was Infused into the lumen at 0.39 ml per min for 20 min and 0.10 ml per min for the final 40 min of the experiments. The small Intestine was then removed and mucosal... 

The enzyme vertebrate collagenase was partially purified from a lyophilized tissue culture medium of back skin from tadpoles. The medium was harvested,... 

Preparation for Bioassav. The standard in vitro assay for allatostatins (1) is a two hour organ culture of corpora allata during which the inhibition of release of radiolabeled JH III from [ H-methyl] methionine is measured accordingly, the test sample must be free from unphysiological contaminants before incorporation into the modified tissue culture medium 199 (1). Our preparative-scale procedures all involved volatile modifiers (hydrochloric acid, formic acid, ammonium formate, TFA) which could be removed along with the solvents (ethanol, water, acetonitrile) this analytical workup of aliquots for bioassay was the only occasion when we used vacuum evaporation. 

Volatile solvents and solutes, up to 0.3 ml, were removed by room temperature centrifugal vacuum (SpeedVac, Savant) in siliconized tubes pre-loaded with 200 pg BSA and 30 fig bacitracin. We found that trace amounts of mercaptoethanol seriously elevate the blanks in the standard radiolabeled isooctane partition assay (13), and residues of TFA require careful readjustment of the pH of the tissue culture medium. Therefore, evaporation was routinely extended for approximately 30 minutes beyond the time of apparent dryness to ensure good removal of solvent modifiers. Dried residues were dissolved in 0.85 ml of modified culture medium 199 (1) by one minute of ultrasonication through the walls of the plastic tube, then left to stand for ca. 30 minutes and finally vortex mixed. 

The choice of receptor fluid can influence the outcome of the study considerably (Ramsey et al., 1994 Bronaugh, 1995). In order to avoid underestimation of skin absorption, the test compound should be soluble in the receptor fluid. On the other hand, the receptor fluid should not damage the barrier properties of the skin membrane. Various receptor fluids have been used, including saline (for hydrophilic test substances) and water/ethanol mixtures, or saline supplemented with bovine serum albumin or poly(ethylene glycol) 20 oleyl ether (for testing of lipophilic compounds). When performing studies with metabolicaUy active skin preparations, the receptor fluid should support the viability of the skin. In these cases, a tissue culture medium is normally used. 

For cells in culture the dish is washed with ice-cold wash solution three times to ensure that all of the protein in the tissue culture medium is removed. Care should be taken to remove all the wash solution in the final wash, after which lysis... 

Procedure. An aliquot of the neutralized diazotized [ I]ISA is added to a washed, buffered suspension of cells (pH 7.5) at 5°. The reaction goes to completion within 15-30 min depending on the cells being labeled. The reaction is stopped by washing the cell suspension 3 or 4 times with a lOx volume of 0.15 M NaCl containing % bovine serum albumin or serum-containing tissue culture medium (e.g., RPMI 1640 with 10% fetal calf serum). 

The assay can be conveniently done in glass or plastic petri plates (60 mm) containing about 4 ml of 0.8% agarose in complement buffer. Guinea pig complement is readily available commercially and generally does not require absorption. Lymphoid cells are prepared by teasing spleens or lymph nodes in cold tissue culture medium buffered with Hepes. If the indirect plaque assay is to be done, an antiserum specific for IgG of the antibody-secreting cells is also required. 

The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 10 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100 /U.1) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a rosette or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted. 

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