Isolation, culture and flow cytometry of bone marrow B cell progenitors.

Isolation and culture of Bone Marrow B cells with IL-7 and CXCL12

MACs column enrichment is better than Sorting (cells does not grow that well compared to column purification).Follwoing is the procedure for isolation of cells from a single mouse. (All steps should be performed on ice unless otherwise mentioned)

1) Isolate the bone marrow cells. Wash with media ((OPTI-MEM+10% FCS+ 1% P/S+1% non-essential amino acids+ 0.1% beta ME)  

2) Perform the ACK lysis. Wash with media PBS (with 2-3% FBS). [ Centrifuge 5min at 1400 rpm at 4 degree)

3). Throw out supernatant and resuspend the cells in 1ml media.

4) Stain with either B220-biotin/CD19-biotin (I prefer B220) antibody (30 min on ice) in 1ml media (OPTI-MEM+10% FCS+ 1% P/S+1% non essential amino acids+ 0.1% beta ME) . Wash (PBS ph7.2+ 2% FCS).

5) Resuspend in 400 micro liter of MACs buffer (PBS pH 7.2+0.5% BSA + 2mM EDTA..always keep in cold..never warm to RT)

5) Then add Streptavidin microbeads (50 micro liter). Keep 30min on ice.

6) Purify by MACs LD column (as per the guideline provided).

7) Around 5-6 million cells would be obtained after enrichment (from 1 mouse).

8) This will contain all the B cells subpopulations (pre-proB, proB, large pre-B, small pre-M, Immature B and recirculating mature B)

9) Wash with PBS.. Spin down... resuspend in 2ml media (see above)...put it in 2 wells (1 ml in each well) of a 24well plate/dish. 

10) Add recombinant IL-7 (20ng/ml) in each well. Put at 37 degree cell culture incubator overnight.

11) After 24 hours look at the cells. It should look healthy and round (without black spots inside).

Please note that there would be lot of dead cells too. Those are mostly the Small pre-B, Immature and mature B. Because IL-7 favors growing of the pro-B and large pre-B population mostly.

12) spin the plate (1400 rpm), take out the media (leaving around 200 micro litter in the plate so that you do not disturb the bottom of the plate.

Add again 1 ml media with 20 ng of IL-7/ml (I each well)

13) Repeat the same after 48 hrs (day 2). In this day, you can see the cells are proliferating (chunk/clustering of cells). If you think the cells are overcrowded split them in two wells (each 1 ml with 20 ng of IL-7).....(pipette in and out and transfer 500 microliter to the next well). Now try have 4 wells. If you think the cells are not overcrowded wait for day 3 to do the same. 

14) Need to continue this culture for 5 days. from Day 3 concentration of IL7 should be 16ng/ml.

15) After 5 days, almost all the cells (around 90%) are in proliferating pre-B cells (large pre-B). (You can check pre-BCR expression. (There won't be any  Immature of mature B cells..all the cells would be IgM negative).

16) If you want to check differential to small pre-B, or eventually to immature B cells, 

Then after 5 days

17) There would be an additional 2 days of culture.

Use 0.2ng/ml of IL-7 (remember, in all the paper the term "NO IL-7" is used; In reality 0.1 to 0.2ng of IL7/ml was used) and 200ng of recombinant CXCL12/ml for 24 hrs.

Chang the media after 24 hrs in a similar way described before with 0.2ng/ml of IL7 and 200ng/ml of CXCL12.

18) After 48 hrs, use the cells for you experiments/assays etc. (NOTE: A boost with addition of 100ng CXCL12 can be done just 3 before taking out the cells for experiment)