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Materials

  • Media Formulation
    • Iscove’s Modified Dulbecco’s Medium (IMDM) Basal Media
    • 10% FBS
    • 2 mM L-glutamine
    • 1X Hypoxanthine-Thymidine (Invitrogen)
    • 50 µM 2-mercaptoethanol
  • Recombinant Murine IL-3
  • Recombinant Murine SCF
  • Culture Flasks or Plates

Procedure

  1. Thaw mast cells following standard procedure and wash once.
  2. Adjust cell concentration to ≥200,000 cells per mL.
  3. Add IL-3 to the medium at 100 U/mL and SCF at 50 ng/mL.
  4. Culture cells in a standard T75 flask at 37°C, 5% CO2.
  5. Cells grow in suspension and may form cell clusters.
  6. Split cells when the culture reaches a density of 3-5 x 106 cells per mL. The medium may appear orange-yellow.
  7. Pipet the culture medium up and down vigorously to break up the cell clumps. Adjust cell concentration by adding fresh medium containing cytokines.

Notes

  • Cells grown as described above can be maintained for at least 3 weeks and will proliferate. During this time expression of CD117 will increase.