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Materials

  • Medium: RPMI 1640 supplemented with 10% fetal calf serum
  • B-LCL

These immortalized human B cells are easy to grow. This protocol gives you the preferred medium and culture conditions that will ensure success.

Procedure

  1. Thaw and count cells following standard procedure.
  2. Adjust cell concentration to ≥200,000 cells per mL.
  3. Add cells to culture flask and incubate at 37°C, 5% CO2.
  4. Cells grow in suspension and may form large cell clusters.
  5. Split cells when the culture reaches a density of 1 x 106 cells per mL. The medium may appear yellow.
  6. Pipet the culture medium up and down vigorously to break up the cell clumps. Remove 80% of the culture and replace with fresh medium.
  7. Cells that have been removed can be cryopreserved or discarded.
  8. Cultures grown in 10% FCS can usually be split 1:5 every 3–4 days. This may vary depending on the lot of FCS used. Maintain the cultures between 2 x 105 and 1 x 106 per mL. Cells can tolerate higher densities, but viability will decline. At cell densities below 200,000 per mL, the cell growth slows significantly.

Notes

  • Some cultures accumulate cell debris that resembles bacterial contamination. This material is actually a bit larger than bacteria and does not have the same Brownian motion.
  • B-LCL express CD19, HLA-DR and CD86.