ML-DmBG3-c2

Source: DGRC, stock #68. 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 the lot that has been reserved for modENCODE.

Note: MITILS has requested that users of cell lines made at MITILS file a voluntary compliance agreement by signing and sending a copy to their representative. Note that the agreement covers only the laboratory whose PI and institutional officer have signed the document.

Medium

Schneider's medium + 10% heat-inactivated FCS + 10 μg/ml insulin.

Note: BG3-c2 cells are very sensitive to the lot of FCS. 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 1:1000 in medium + FCS; the complete medium is good for at least several months at 4° C.

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].

ML-DmBG3-c2 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; BG3-c2 cells are surface-adherent, but can be easily 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. Dilute the resulting cell suspension in fresh medium, and dispense to fresh tissue-culture grade Petri plates. BG3-c2 cells are very sensitive to over-dilution, and will not grow well at concentrations less than around 2-3X10⁶ cell/ml. A culture should be scaled up when the cells cover most of the surface and are beginning to form dense clumps; at this point, they can be diluted approximately 3-4 fold. The line is quite slow-growing; it will only need to be transferred every 4-7 days.
• For experiments, use the cells at mid-exponential growth, approximately the concentration shown in this photo on the DGRC web-page or a little higher.
  
  

  Note:This line is quite delicate; 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).

  
  
  

  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 BG3-c2 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.