CME W1-Cl.8+
Source: DGRC, stock #151. When ordering, please also send an e-mail to the DGRC (dgrc@indiana.edu) stating that you are ordering these cells for modENCODE; this will ensure that you receive cells from the lot that has been reserved for modENCODE.
Medium
M3 medium + 2% heat-inactivated FCS + 5 μg/ml insulin + 2.5% fly extract.
- Note:Cl.8 cells are not very sensitive to the lot of FCS. Nonetheless, we recommend that everyone use the same lot of serum (see below) to maximize consistency within modENCODE.
- 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 (https://dgrc.bio.indiana.edu/include/file/tissue-culture-medium-additions.pdf). You may make the extract yourself, or order it from the DGRC.
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 [https://dgrc.bio.indiana.edu/include/file/ThawingCells.pdf].
Cl.8 cells recover slowly after thawing. Once they reach approximately 107 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 2-3x106 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 this photo on the DGRC web-page or a little higher.
- Note:This line is less robust than Kc or S2; it is unlikely to recover if it is allowed to get too dense, or if it is over-diluted. If this happens, we recommend going back to a frozen stock (see below).
- 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.
- Note: It is a good idea to freeze some cells for back-up use as soon as you have the cells growing well in your lab (for protocol, see https://dgrc.bio.indiana.edu/include/file/FreezingCells.pdf). We leave it to the individual investigator whether to use your own frozen stock to replenish your stock as necessary, or whether to order more cells from the DGRC. The DGRC is putting aside a sufficiently large stock of a single lot that we anticipate being able to supply several ampoules to each modENCODE user should that be necessary.