S2-DRSC
Source: DGRC, stock #181. 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 lot 181A1, the lot that has been reserved for modENCODE. This lot is now available.
- Note: These are the S2 cells that have been used extensively for RNAi screens at the DRSC. They were recently given to the DGRC for distribution.
Medium
Schneider's medium + 10% heat-inactivated FCS.
- Note: S2 cells are not particularly sensitive to the lot of FCS. We recommend that everyone use the same lot of serum (see below) to maximize consistency within modENCODE, but this is less critical than for some of the other lines.
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].
S2-DRSC cells take very little time to begin to grow after thawing. Once they reach approximately 107 cells/ml (enough to cover the surface of the flask), they should be diluted 3-fold as follows: Using a Pasteur pipet, blow medium at the surface of the flask to dislodge the cells; S2-DRSC cells are surface-adherent, but can be easily dislodged by this procedure. Add 10 ml fresh medium to the 5 ml in the flask. Gently mix by pipetting up and down. Transfer 10 ml of the cell suspension to a 10 cm plate. The 5 ml remaining in the flask can be used as a back-up culture. It will take 1-2 weeks 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 by simply diluting a cell suspension in fresh medium, and dispensing to fresh tissue-culture grade Petri plates. In order to remove the cells from the surface of the plate, blow medium at them, using a sterile Pasteur pipet or a sterile serological pipet. Cells should not be allowed to grow to higher concentrations than 107/ml; this can be easily estimated as the point at which the cells would cover the surface of the plate in a single layer if they were actually surface-adherent (which they aren't). They should be diluted to a concentration not less than 106/ml. After thawing, it will probably take a few transfers before the cells reach their normal growth rate; at that point, they will need to be transferred approximately once or twice a week.
- For experiments, use them in mid-exponential growth (around 5X106 cells/ml; at this concentration, the cells cover about half of the surface of the plate). (For calibration, this photo on the DGRC web-page shows cells at about 5-6x106 cells/ml.
- Note: This line is pretty rugged, and will recover if allowed to over-grow for a few days, or if slightly over-diluted. However, this should be avoided, in the interests of making sure that the cells used are similar in all of the modENCODE labs.
- Note: These cells can be grown in T flasks and in spinner cultures. However, their properties change with the culture vessel. To maximize consistency within modENCODE, they should be grown in 100 mm tissue-culture grade Petri plates (Corning 430167).
- Do not grow S2-DRSC continuously for more than 6 months. This is a pretty stable line, but in order to avoid the accumulation of small changes, it is good practice to go back to a frozen stock after a few months, even if the culture looks healthy. 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.