Culture of B-Lymphoblastoid Cell Lines

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

Materials

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

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.