Download Methods in Cell Biology, Vol. 5 by David M. Prescott (Ed.) PDF
By David M. Prescott (Ed.)
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Additional info for Methods in Cell Biology, Vol. 5
Example text
0. For preparing lower concentrations of SSC, simply dilute the 1OX solution with distilled water. 1. PROCEDURES FOR MAMMALIAN CHROMOSOME PREPARATIONS 13 11. HYPOTONIC SOLUTION Hypotonic solutions can be prepared in a variety of ways. 0%sodium citrate solution is very good for direct bone marrow preparations as well as for many cell cultures. However, when cells are removed from culture flasks by trypsin or pronase solutions, the cells sometimes clump if citrate solution is used. A diluted growth medium (growth medium diluted two or three times with distilled water) will eliminate clumping.
HSU 2. Transfer the tissue fragments to a culture vessel. The T-series or plastic flasks are ideal because they have good optical quality for observation of cellular growth. However, screw-capped medicine bottles can be used as substitutes if T-flasks are not available. Add a small amount of growth medium to each flask. Gas the flasks with 10%CO, and stopper tightly. Whenever feasible, set up a duplicate culture. 3. Add a small amount of growth medium to each flask the next day. Thereafter, feed the cultures every other day or at least three times per week.
Sealed ampoules should remain at room temperature for approximately 2 hours in order to allow equilibrium of glycerol between the cells and the medium. e. Six ampoules can be placed in a powder box, which is in turn placed in a compartmentalized metal or plastic rack inside the dry-ice chest. 2. THAWING a. Remove the ampoule from the freezer and immediately place it in a beaker containing warm water (temperature not more than 37°C). Shake the ampoule rapidly until the medium is thawed. b. Open the ampoule aseptically and add 1 ml of fresh medium with a Pasteur pipette.