1. Collect bone marrow from 2-6 bones/mouse.
2. Purify B220+ cells through MACS positive selection using anti-B220-microbeads.
We usually obtain 10% of our total bone marrow as B220+ (around 2-4E6 cells from one mouse) and we culture all of it.
3. Wash cells with OptiMEM and culture cells in OptiMEM with 15% FBS, pen/strep, L-glutamine, BME, and 5ng/mL IL-7 at a concentration of at least 2E6 cells/ml.
I have never had a problem with culturing cells more concentrated than 2E6cells/mL as long as I check the cells every 2 days, but once the concentration dips below 2E6/mL, I start to see a lot of cell death.
We buy Hyclone SH30071.03 serum for our bone marrow cultures. It is more expensive than our normal culture serum, but pre-B cell survival is greatly enhanced in it. Other labs have mentioned that the type of serum greatly effects the survival of cells in pre-B cultures.
4. Culture for 2 days, collect and count. Spin cells down at 350g.
The cell count does not change much from the day of collection due to proliferation of pre-B cells counter balanced by the death of immature and mature B cells. Here you remove a lot of the dead B cells.
5. While cells are spinning, dilute viral supernatent in half with OptiMEM and add polybrene to a final concentration of 8ug/mL. Let sit on ice for 10 minutes.
6. Resuspend cells in viral supernatent. Transfer to plate for spinfection. Spin plate at 927g for 90 minutes and then place back in incubator for 60 minutes.
7. For spinfection, 3-4 mL of cells can be placed in one well of a 6 well plate, 2-3 mL in a 12 well plate, and 1-2 mL in a 24 well plate.
8. Collect cells out of plate after incubation, spin cells to wash out viral supernatent, and resuspend cells at 2E6/mL in culture conditions for pre-B cells as above.
9. Continue culturing cells for up to 3 weeks, changing media every 2-3 days.
For multiple infections, viral supernatants can be mixed and one spinfection can be performed, or sequential spinfections can be done. Sequential spinfection leads to decreased infectivity of cells, and after the first spinfection, infection efficiency drops.
*To increase rate of proliferation and to purify pro-pre B cells, 10ng/ml of SCF can be added to cultures as well.