3.2 Meningococcal
Growth, Fixing,
and Staining
1. Streak a loopful of meningococcal frozen stocks on blood agar.
Incubate for 14–16 h at 37
C, 5% CO 2 .
2. Harvest cells from the overnight growth into 10 mL bacterial
fixing solution using a sterile 10 μL disposable loop.
3. Incubate for !4 h at RT.
4. Wash meningococcal cells thrice in 10 mL 0.1 M NaHCO 3 .
Resuspend cells to OD 600 of 0.1 (~10
7 cells/mL).
5. Resuspend each aliquot of 10
7 cells in 495 μL of 0.1 M
NaHCO 3 . Add 5 μL of cell staining solution. Incubate for
1 h at 37
C.
6. Wash stained meningococcal cells at 800 Â g for 10 min in
500 μL of 0.1 M NaHCO 3 .
7. Repeat step 6 above four more times.
8. Resuspend stained meningococcal cells in ICA buffer. Stained
cells can be stored overnight at 4
C, protected from light.
3.3 ICA
3.3.1 PBMC Thawing
1. Warm the RPMI-1640 and R10 to RT. Transfer 9 mL of
RPMI-1640 per five million live PBMCs into a 50 mL
centrifuge tube.
2. Transfer PBMC vials from À80
C to a 37
C water bath.
Check the vials periodically and remove following complete
thawing.
3. Immediately after complete thawing, add the PBMC sample
dropwise into the RPMI-1640 medium.
4. Pellet the PBMCs at 500 Â g, RT for 8 min. Discard the
supernatant.
5. Resuspend the PBMCs in 10 mL of ICA buffer.
6. Add 10 μL of the sample to 10 μL of trypan blue dye. Count
the number of cells in this mixture using an automated cell
counter.
7. Pellet the PBMCs (from step 5 above) at 500 Â g, RT for
8 min. Discard the supernatant.
8. Resuspend the PBMCs at a concentration of 3–4 million live
PBMCs per 100 μL in ICA buffer. Store on ice.
3.3.2 IgG Secretion
and Capture
1. Label 2 mL microcentrifuge tubes as follows:
(a) “C” for unstained PBMCs.
(b) “CS” for stained PBMCs (no ICA).
(c) “CF-APC” for fluorescence minus APC control.
(d) “CF-BV421” for fluorescence minus BV421 control.
(e) “CF-BV605” for fluorescence minus BV605 control.
(f) “CF-PE” for fluorescence minus PE control.
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Sara Siris et al.
Growth, Fixing,
and Staining
1. Streak a loopful of meningococcal frozen stocks on blood agar.
Incubate for 14–16 h at 37
C, 5% CO 2 .
2. Harvest cells from the overnight growth into 10 mL bacterial
fixing solution using a sterile 10 μL disposable loop.
3. Incubate for !4 h at RT.
4. Wash meningococcal cells thrice in 10 mL 0.1 M NaHCO 3 .
Resuspend cells to OD 600 of 0.1 (~10
7 cells/mL).
5. Resuspend each aliquot of 10
7 cells in 495 μL of 0.1 M
NaHCO 3 . Add 5 μL of cell staining solution. Incubate for
1 h at 37
C.
6. Wash stained meningococcal cells at 800 Â g for 10 min in
500 μL of 0.1 M NaHCO 3 .
7. Repeat step 6 above four more times.
8. Resuspend stained meningococcal cells in ICA buffer. Stained
cells can be stored overnight at 4
C, protected from light.
3.3 ICA
3.3.1 PBMC Thawing
1. Warm the RPMI-1640 and R10 to RT. Transfer 9 mL of
RPMI-1640 per five million live PBMCs into a 50 mL
centrifuge tube.
2. Transfer PBMC vials from À80
C to a 37
C water bath.
Check the vials periodically and remove following complete
thawing.
3. Immediately after complete thawing, add the PBMC sample
dropwise into the RPMI-1640 medium.
4. Pellet the PBMCs at 500 Â g, RT for 8 min. Discard the
supernatant.
5. Resuspend the PBMCs in 10 mL of ICA buffer.
6. Add 10 μL of the sample to 10 μL of trypan blue dye. Count
the number of cells in this mixture using an automated cell
counter.
7. Pellet the PBMCs (from step 5 above) at 500 Â g, RT for
8 min. Discard the supernatant.
8. Resuspend the PBMCs at a concentration of 3–4 million live
PBMCs per 100 μL in ICA buffer. Store on ice.
3.3.2 IgG Secretion
and Capture
1. Label 2 mL microcentrifuge tubes as follows:
(a) “C” for unstained PBMCs.
(b) “CS” for stained PBMCs (no ICA).
(c) “CF-APC” for fluorescence minus APC control.
(d) “CF-BV421” for fluorescence minus BV421 control.
(e) “CF-BV605” for fluorescence minus BV605 control.
(f) “CF-PE” for fluorescence minus PE control.
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Sara Siris et al.