P-GSE24307-1 - P-GSE24307-1

Cell Line Growth CME W1 protocol; CME W1-Cl.8+ Source: DGRC, stock #151. M3 medium + 2% heat-inactivated FCS + 5 μg/ml insulin + 2.5% fly extract. * The source of insulin is probably not critical, but should be kept constant in case it matters. Purchase a sterile 10 mg/ml solution of human insulin from Sigma (cat. no. I9278); this can be stored for at least 6 months at 4° C. Dilute 1:2000 in medium + FCS + fly extract. The complete medium is good for about a month at 4° C, with the fly extract being the most unstable component. * Fly extract is prepared according to the protocol given on the DGRC web-site (
). You may make the extract yourself, or you request it from the Cherbas lab. As a courtesy, we will supply fly extract at no cost to members of the modENCODE consortium, but you will be asked to supply a Fedex number to pay for the shipment. You will need 2.5 ml per 100 ml of medium. Tubes of fly extract can be stored for at least 1 year at -20° C. Thawing the tube requires no special precautions. To order fly extract, send e-mail to Lucy Cherbas (dgrc@cgb.indiana.edu) . Thawing cells As soon as possible after you receive them, thaw an ampoule of frozen cells in a 25 cm2 T-flask, precisely as described in the DGRC protocol [1]. Cl.8 cells recover slowly after thawing. Once they reach approximately 10^7 cells/ml (enough to cover the surface of the flask), they should be diluted as follows: Using a Pasteur pipet, blow medium at the surface of the flask to dislodge the cells; Cl.8 cells are the most strongly surface-adherent of the 4 modENCODE lines, but they can still be dislodged by this procedure. Transfer the contents of the flask to a 10 cm plate; add 5 ml of fresh medium to the plate and pipet gently up and down to mix. Add 5 ml of fresh medium to the flask; there are generally enough cells left on the surface that they will readily grow up. Sometimes, after a few days the cells in the plate do not seem as healthy as those in the flask; if this happens, go back to the flask and repeat the procedure. It will take several transfers for the cells to reach their normal growth rate; they should not be used for experiments during that time. General instructions for culture maintenance * Scale up cells as follows: Gently remove the cells from the substrate by blowing medium at the surface of the plate from a serological pipet. Cl.8 cells are more surface-adherent than the other 3 lines in the modENCODE collection, but they can still be dislodged in this way. Dilute the resulting cell suspension in fresh medium, and dispense to fresh tissue-culture grade Petri plates. Cl.8 cells are sensitive to over-dilution, and will not grow well at concentrations less than around 2-3x10^6 cells/ml. A culture should be scaled up when the cells cover the entire surface and are starting to pile up; at this point, they can be diluted approximately 5-fold. The line grows more slowly than Kc or S2; it will need to be transferred about once or twice a week. * For experiments, use them at mid-exponential growth, approximately the concentration shown in the photo on the DGRC web-page (
) or a little higher. * Do not grow Cl.8 continuously for more than 4 months. This line is distinctly less stable than the other three lines. Go back to a frozen stock earlier if the cells look unhealthy.
Experiment E-GEOD-24307