General comments on culturing cells in vitro

General comments about S2R+ cells

  1. established from late stage embryos (20-24 h), Oregon R strain
  2. macrophage like lineage (hemocyte like)
  3. S2 cells and S2R+ cells with the later to be much more adherent. (Schneider (1972) J. Embryol. Exp. Morphol. 27, 353-365, Yanagawa et al. (1998) JBC 273, 32353-32359)
  4. other cells lines are described at the Bloomington web site.

Describtions of general procedures:

Please find here a decription of general and transfection procedures (from Current protocols).
Please find here the protocols from Cherbas at Bloomington.

Culture conditions:

  1. for slow growth: 22-25¡C, for regular growth: 28¡C; without CO2
  2. grow in Schneider's Drosophila medium (commercially available) or in M3 + BPYE, both supplemented with 10% heat inactivated (or in Sf-900 II SFM medium) - heat inactivation should happen for 30-60 min to a bottle having room temperature - M3 is made using 500 ml of commercially available Shield's and Sang M3 medium + addition of bactopeptone (1 g/l) and yeast extract (2.5 g/l)
  3. Fly extracts can be added if necessary.
  4. Insulin (human or bovine) can be added: dissolve 10 mg in 0.25 ml of 0.01 N HCl, dilute in 10 ml medium, filter-sterilize and store aliquots at -20¡C.
  5. Conditioned medium can be used to help cells growing. Use a 0.22 µm filter before adding to cells.
  6. Drosophila PBS for living cells is 120 mM NaCl in phosphate buffer at pH 6.7
  7. Cells are healthy in the range of 10^6 - 10^7 cells/ml, can be passaged by simple resuspension and dilution to 1:3 to 1:5, use medium pre-warmed for at least 15 min. However, cells will not grow when seeded at a density below 5 x 105/ml.

Freezing/Thawing:

  • version A of thawing is to prepare a 25cm2 flask with 5 ml medium, add cells as soon as they are thawn (hand-warmed) and incubate for 1-2 h. Then, change medium and again 24 h later. version B is to use a 15 ml Falcon tube with 10 ml medium, add the cells, spin for 3 min at 200xg and discard the medium. Then, the cells are resuspended in fresh medium and seeded in a 25cm2 flask.
  • Typically freeze about 0.5 ml of a 2 x 107 cells/ml suspension, using either FCS with 10% DMSO or medium supplemented with 20% FCS and 10% DMSO.