Tissue culture sterilization

Buffer filtration device (either a glass filtration unit, fitted with a 0.45-itm membrane, connected to a side-arm flask or a tissue culture sterilization filter unit). 

Hanks Solution. For tissue culture (sterile, phenol red added as pH (indicator) (Sigma H8389). This replaces the more expensive MEM (minimal essential medium). 

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 The tissue-culture supernatant is withdrawn from the tissue-cnlture flask and filtered through a 0.22-p filter to ensure sterility.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. 

Polyarylsulfones offer materials with good thermal-oxidative stability, solvent resistance, creep resistance, and good hydrolytic stability. Their low flammability and smoke evolution encourage their use in aircraft and transportation applications. They hold up to repeated steam sterilization cycles and are used in a wide variety of medical applications such as life support parts, autoclavable tray systems, and surgical and laboratory equipment. Blow-molded products include suction bottles, surgical hollow shapes, and tissue culture bottles. PPS has a number of automotive uses including as an injection-molded fuel line coimector and as part of the fuel filter system. 

A sterile plastic box that can accommodate six 100 ml tissue culture plates Microcentrifuge tubes Sterile pipette tips Microscope... 

Detach the CLL cells by gently pipetting up and down using a 1,000 pi pipette tip. Rotate the dishes while pipetting to detach the CLL cells all around the tissue culture dishes. Collect the media containing cells in an appropriate sterile tube. Examine the tissue culture dishes under the microscope to ensure that all the CLL cells are collected and the layer of fibroblasts is still intact (see Note 9) (Fig. Id). 

WT N1H3T3 cells are more prone to detach than CD154 N1H3T3, so detaching CLL cells from the WT N1H3T3 cells requires extra care. If discontinuities or ruptures in the layer of fibroblasts are noticed, CLL cells may be seeded at this step in 60 mm culture dishes for 2 h to allow possible NIH3T3 cells present in the sample to reattach on the surface of the tissue culture dishes. The tissue culture dishes are then incubated in a humidified atmosphere of 5% CO2 at 37°C. Afrer incubation, examine the tissue culture dishes for the presence of adherent cells and carefully collect the CLL cells in an appropriate sterile tube. 

Waller, G. R. and C. F. Cumberland. High production of caffeine by sterile tissue cultures of Coffea arabica. Colloq Sci Int Cafe [C.R.] 1980 9 611-618. Koenig, W. A., W. Rahn, and R. Vetter. Identify and quantify emetic active constituents in roast coffee. Colloq Sci Int Cafe [C.R.] 1980 9 145-149. 

Plastid transformation is highly dependent on the tissue culture process because it enables copies of the wild-type plastid genome to be selectively eliminated before plant regeneration (Maliga, 2003). However, many of the crop species regenerated in this way turn out to be sterile, a consequence of plant regeneration from tissue culture. As mentioned earlier, the transformation of Arabidopsis, tomato, potato, rice, and rape seed oil has been achieved at very low efficiencies, and the resulting transformants... 

Microorganisms in liquids and gases can be removed by microfiltration hence, air supplied to aerobic fermenters can be sterilized in this way. Membrane filters are often used for the sterihzation of liquids, such as culture media for fermentation (especially for tissue culture), and also for the removal of microorganisms from various fermentation products, the heating of which should be avoided. 

Although Brevundimonas (Pseudomonas) diminuta (ATCC 19146) is most commonly used for sterilizing-grade filter validation, in certain applications other bacteria are used. For example, when it is necessary to demonstrate removal of mycoplasma in applications involving sera and tissue culture media, membranes having a smaller pore size rating, eg, 0.1 Jim, are frequendy used. For these membranes, Picholeplasma laidlawii may be employed for validation purposes (9). 

Media basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use. 

Costar 25-cm2, 75-cm2, and 225-cm2 vented sterile tissue culture flasks (cat. nos. 3056, 3376, 3001 Coming Incorporated). 

In 1907, Ross Granville Harrison introduced tissue culture as a new technique for the study of nerve fibre outgrowth [24], At that time, it was hardly envisioned that cell culture would become the most widespread research tool in life sciences and an important method for preparing antibodies, vaccines and drugs. During the development of tissue culture, parameters such as sterility, temperature, gas mixture, medium composition and substrate features were found to be critical for... 

Cells should be dispersed as described in 7.1.1, but the culture vessel should contain many small fragments (0.05 cm2) of coverslips to which the cells will attach and grow. Using sterile forceps remove coverslips fragments on which single cells have attached (check this microscopically) and transfer them to separate wells in a tissue culture tray ( 3.2). 

In the appropriate culture medium, tissue explants give rise to callus tissue. Callus tissue is comprised of large, thin-walled parenchyma cells. It is similar to the undifferentiated tissue produced by plants as a repair mechanism when they are injured. In tissue culture, dedifferentiated callus can be induced to form plantlets that grow into normal plants. The induction of callus occurs when a sterile explant is brought into contact with a nutrient medium, which contains substances that initiate cell division and support growth. An explant may be a uniform piece of tissue or tissue derived from different cell types (Yeoman, 1973). Storage parenchyma tissue from Jerusalem... 

Sterile, endotoxin free, tissue culture-grade water. 

Take the femurs to the tissue culture area, wipe them again with 70% alcohol, and place them in a sterile Petri dish containing 5 mL of Fischers medium with 20% DHS. 

Standard tissue culture facilities, including sterile hood. 

Sacrifice mouse and aseptically remove the spleen to a 110-mm2 tissue culture dish containing sterile serum-free DMEM without HEPES. All subsequent steps should be carried out in a tissue-culture hood. 

Tissue culture filter unit, 500 mL, 0.2-p cellulose acetate, sterile Nalgene... 

Clear, sterile, tissue culture treated multi-well plates (6-well, 12-well, or 24-well, etc.)... 

All solutions must be made up in tissue culture grade water (Milli-Q or equivalent). Solutions 4, 5, 9, 10, 21, 24-2 7, 29, and 36 are stored at -20°C and 11,14,17,23,28,30 and 34 are stored at 4°C. Solutions 18,19, and 31 are made immediately before use. The remaining solutions are stored at room temperature. All solutions used with the cultured cells must be kept rigorously sterile and all manipulations must be performed in a sterile flow hood. 

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