Kc167

Source: DGRC, stock #1. When ordering, please also send an e-mail to the DGRC (dgrc@iu.edu) stating that you are ordering these cells for modENCODE; this will ensure that you receive cells from lot 001A3, 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:This line grows well in the serum-free medium CCM-3, and we have used it extensively for experiments in this medium. However, the ploidy of Kc167 is somewhat unstable in CCM-3, and the cells tend to be pseudo-tetraploid. We therefore recommend that the modENCODE consortium use M3+BPYE+10% FCS, in which the cells are stably pseudo-diploid.

Note:Kc 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], with the following modification: Because these cells adhere very weakly to the substrate in the medium that we are using for the modENCODE project, it is advisable to spin the cells to remove the DMSO-containing medium rather than just removing the medium after letting the cells settle. Add the frozen cells to 5 ml of medium as described in the protocol. Then transfer the cells to a sterile centrifuge tube and spin at approximately 1500 g for 2 minutes to pellet the cells. Discard the supernatant, and resuspend the cells in 5 ml of fresh medium, and transfer the suspension to a fresh 25 cm² T flask. It is not necessary to repeat this procedure. .

Kc cells take almost no 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: Add 10 ml fresh medium to the 5 ml in the flask. Gently disperse the cells by pipetting up and down; Kc167 grown in the medium that we have specified are only very slightly surface adherent, and can very easily be dislodged from the substrate. 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. 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. (For calibration: this photo on the DGRC web-site shows a culture at about 1.5X10⁶ cells/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 every 2-3 days.
• For experiments, use them in mid-exponential growth (with the cells covering about half of the surface of the plate).
  
  

  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 Kc167 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.