These immortalized human B cells are easy to grow. This protocol gives you the preferred medium and culture conditions that will ensure success.
- Thaw and count cells following standard procedure.
- Adjust cell concentration to ≥200,000 cells per mL.
- Add cells to culture flask and incubate at 37°C, 5% CO2.
- Cells grow in suspension and may form large cell clusters.
- Split cells when the culture reaches a density of 1 x 106 cells per mL. The medium may appear yellow.
- Pipet the culture medium up and down vigorously to break up the cell clumps. Remove 80% of the culture and replace with fresh medium.
- Cells that have been removed can be cryopreserved or discarded.
- 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.
- 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.