11. Immediately prior to use, pass the EB suspension through a
27 G needle 5–10 times to break up any large aggregates.
3.3 Staining
of Memory B Cells
1. Thaw cryopreserved PBMCs by placing the frozen tube
directly into a 37
C water bath until almost all of the sample
has thawed.
2. Transfer the entire sample to a 50 mL centrifuge tube and top
up to 30 mL with room temperature R0 media. Add the media
dropwise to the cell suspension using a serological pipette while
gently rocking the tube to mix the contents.
3. Centrifuge the cell suspension for 5 min at 500 Â g at 4
C.
4. Pour off the supernatant and resuspend the cell pellet in 45 mL
of R0. Centrifuge the cell suspension for 5 min at 500 Â g at
4
C.
5. Pour off the supernatant and resuspend the cell pellet in 1 mL
of PBS. Count the cells using any preferred method.
6. Top up the cell suspension to 45 mL with PBS. Centrifuge the
cell suspension for 5 min at 500 Â g at 4
C.
7. Aspirate the supernatant and resuspend the pellet in 100 μL of
PBS. Transfer the cell suspension to a 5 mL FACS tube.
8. Add the recommended volume of viability dye based on the cell
count from step 5. Incubate the cells at room temperature for
20 min in the dark.
9. Add 1 mL of 1% PBS/BSA to the cells. Centrifuge for 5 min at
350 Â g at 4
C and pour off the supernatant.
10. Resuspend the cell pellet in the 100 μL of labeled EB suspension from Subheading 3.2. Incubate the cells at room temperature for 20 min in the dark.
11. Add 1 mL of 1% PBS/BSA to the cells and centrifuge for 5 min
at 350 Â g at 4
C.
12. Prepare a master mix containing titrated volumes of fluorescent
antibodies against CD3, CD14, CD19, CD27, CD38, IgM,
IgD, IgG, and IgA, to a total volume of 100 μL, in 1%
PBS/BSA (see Notes 6 and 7).
13. Pour off the supernatant from the cell pellet and resuspend in
100 μL of the antibody master mix. Incubate for 20 min on
wet ice.
14. Add 1 mL of 1% PBS/BSA to the cells. Centrifuge for 5 min at
350 Â g at 4
C and pour off the supernatant.
15. Resuspend in 500 μL of 1% PBS/BSA. Pass the cell suspension
through a 35 μm mesh to remove cell aggregates prior to
sorting. Keep the cells on ice and in the dark until loading
into the sorter.
24
Christopher L. Pinder et al.
27 G needle 5–10 times to break up any large aggregates.
3.3 Staining
of Memory B Cells
1. Thaw cryopreserved PBMCs by placing the frozen tube
directly into a 37
C water bath until almost all of the sample
has thawed.
2. Transfer the entire sample to a 50 mL centrifuge tube and top
up to 30 mL with room temperature R0 media. Add the media
dropwise to the cell suspension using a serological pipette while
gently rocking the tube to mix the contents.
3. Centrifuge the cell suspension for 5 min at 500 Â g at 4
C.
4. Pour off the supernatant and resuspend the cell pellet in 45 mL
of R0. Centrifuge the cell suspension for 5 min at 500 Â g at
4
C.
5. Pour off the supernatant and resuspend the cell pellet in 1 mL
of PBS. Count the cells using any preferred method.
6. Top up the cell suspension to 45 mL with PBS. Centrifuge the
cell suspension for 5 min at 500 Â g at 4
C.
7. Aspirate the supernatant and resuspend the pellet in 100 μL of
PBS. Transfer the cell suspension to a 5 mL FACS tube.
8. Add the recommended volume of viability dye based on the cell
count from step 5. Incubate the cells at room temperature for
20 min in the dark.
9. Add 1 mL of 1% PBS/BSA to the cells. Centrifuge for 5 min at
350 Â g at 4
C and pour off the supernatant.
10. Resuspend the cell pellet in the 100 μL of labeled EB suspension from Subheading 3.2. Incubate the cells at room temperature for 20 min in the dark.
11. Add 1 mL of 1% PBS/BSA to the cells and centrifuge for 5 min
at 350 Â g at 4
C.
12. Prepare a master mix containing titrated volumes of fluorescent
antibodies against CD3, CD14, CD19, CD27, CD38, IgM,
IgD, IgG, and IgA, to a total volume of 100 μL, in 1%
PBS/BSA (see Notes 6 and 7).
13. Pour off the supernatant from the cell pellet and resuspend in
100 μL of the antibody master mix. Incubate for 20 min on
wet ice.
14. Add 1 mL of 1% PBS/BSA to the cells. Centrifuge for 5 min at
350 Â g at 4
C and pour off the supernatant.
15. Resuspend in 500 μL of 1% PBS/BSA. Pass the cell suspension
through a 35 μm mesh to remove cell aggregates prior to
sorting. Keep the cells on ice and in the dark until loading
into the sorter.
24
Christopher L. Pinder et al.