B cell magnetic fractionation

B cell magnetic fractionation

Immediately after isolating fresh PBMCs from 48 ml of blood or thawing 1 to 3 frozen vials of PBMCs from the same donor obtained form 16 ml of blood each, B cells were isolated with the negative selection Human B Cell Enrichment Kit (STEMCELL Technologies) according to the manufacturer's instructions. 

The purple magnet from STEMCELL and the normal size FACS polystyrene round-bottom tube were used. To this tube we added PBMCs in 1 mL of 1% FBS, the B cell negative cocktail (50 μl/ mL of sample) and mixed it well with up and down pipeting prior to 4ºC incubation for 12-15 minutes. Close to the end of the incubation time, we vortexed the kit’s beads, and when time was up added them to sample and mix (75ul/ml) and incubated 5min at 4ºC. After this the volume was brought to 3,5-4ml with 1% FBS, mixed pipetting up and down, and the tube was inserted in the magnet. Five min later, with the tube still inside the magnet and in one continuous motion, we inverted the magnet and tube, pouring the enriched B cell suspension into a new tube. 

Following B cell enrichment, the cell suspension was centrifuged to bring the cell density to a volume allowing antibody staining and B cells were fractionated into atypical MBCs (CD19⁺CD21⁻CD27⁻), classical MBCs (CD19⁺CD27⁺CD21⁺) and naïve B cells (CD19⁺CD21⁺CD27⁻) using a three-step magnetic bead-based selection procedure. B cells were first incubated with 1:50 dil of PE-conjugated anti-CD21 mAB (HB5) (eBioscience), in 1% FBS and incubated for 30min at 4ºC, followed by Anti-PE MultiSort MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions to separate CD21⁻ and CD21⁺ B cells. MS collums and and OctoMACS magned were used. 

After staining the cells qwith the anti-CD21 PE antibody, they were washed and incubated with Anti-PE MultiSort MicroBeads using the “intermediate” recommendation of 90ul of buffer and 10ul of Anti-PE MultiSort MicroBeads, mixed well and incubated for 15 minutes at 4°C, and then passed on the MACS Column and MACS Separator magnet. Following positive selection and after washing the column 3x, both fractions were recovered.

The magnetic particles were removed from the CD21⁺ fraction using the MultiSort Release Reagent adding 20 μL MultiSort Release Reagent per 1 mL cell suspension, mixing well and incubating for 10 minutes at 4°C. Then cells were washed, the supernatant completely discarded and cells resuspend cells in 50 μL plus 30 μL of MultiSort Stop Reagent and mixed well.

Finally, CD21⁻ and CD21⁺ B cells were incubated separately with anti-CD27 MicroBeads (Miltenyi Biotec) in 1% FBS according to the manufacturer's instruction to separate CD27⁻ and CD27⁺ B cells. In either case MS collums and OctoMACS magned were used. Cell pellets were resuspended in 80 μL of 1%FBS and 20 μL of CD27 MicroBeads were added and mixed well prior to a 15minutes incubation at 4°C. After incubation cells were washed and resuspended in 500 μL of buffer and passed on the MS columns, and washed 3x with 500ul.

From the CD21⁺ fraction both CD27⁻ and CD27⁺ were recovered. From the CD21⁻ fraction only the CD27⁻were recovered, and hence we obtained naïve B cells (CD19⁺CD21⁺CD27⁻), classical MBCs (CD19⁺CD21⁺CD27⁺) and atypical MBCs (CD19⁺CD21⁻CD27⁻).

The purity of each fraction was verified by flow cytometry using fluorescently labeled Abs specific for CD19 PerCP-Cy5.5 (SJ25C1) dil 1:50, CD21 PE (HB5) (eBioscience) dil 1:50 and CD27 APC (O323) (invitrogen, Life Technologies, Carlsbad, CA) dil 1:20. This method produced 10⁵ to 10⁶ highly pure naïve B cells, classical and atypical MBCs depending on the starting volume of blood.