(f) Anti-CD38 PerCP/Cy5.5: 2 μL.
(g) Anti-human IgA Alexa Fluor 594: 5 μL.
(h) Anti-human IgD BV 786: 5 μL.
(i) Anti-human IgM BV 650: 5 μL.
(j) Zombie NIR Live/Dead: 1 μL.
3. Incubate the single-stain control tubes on ice for 20 min.
4. For the fluorescence minus one controls, add all antibodies
except the APC antibodies to tube “CF-APC” (see step 1 of
Subheading 3.3.2). Repeat this procedure for other tubes
excluding the relevant antibody for each control. For gating
purposes, add all antibodies to tube “CS.” Incubate all control
tubes on ice for 20 min.
5. Wash all bead and PBMC control tubes with 2 mL ICA buffer.
6. Pellet stained beads and PBMCs at 500 Â g, RT for 8 min.
Discard the supernatant.
7. Resuspend stained beads and PBMCs in 750 μL of ICA buffer.
Transfer these into fresh, labeled 5 mL polystyrene tubes. Store
on ice, in dark, until flow cytometry processing.
After IgG secretion and capture, transfer the sample into a fresh,
labeled (“CS-ICA”) tube and proceed with the following steps:
1. Pellet PBMCs in tube “CS-ICA” at 500 Â g, RT for 8 min.
Discard the supernatant.
2. Resuspend PBMCs in tube “CS-ICA” in 100 μL ICA buffer.
3. Add FAM SE-stained bacteria (at a ratio of 16 bacterial cells to
1 PBMC—see Note 6) to CS-ICA. Incubate at RT, in the dark,
for 20 min.
4. Wash PBMC–bacteria complexes in tube “CS-ICA” with 2 mL
ICA buffer.
5. Pellet PBMC–bacteria complexes at 500 Â g, RT for 8 min.
Discard the supernatant.
6. Resuspend PBMC–bacteria complexes in 74 μL ICA buffer.
7. Prepare a cocktail of all fluorescent antibodies as follows:
(a) Anti-CD3 APC: 2 μL.
(b) Anti-CD14 APC: 1 μL.
(c) Anti-CD19 BV 421: 2 μL.
(d) Anti-CD20 BV 605: 3 μL.
(e) Anti-CD27 PE: 1 μL.
(f) Anti-CD38 PerCP/Cy5.5: 2 μL.
(g) Anti-human IgA Alexa Fluor 594: 5 μL.
(h) Anti-human IgD BV 786: 5 μL.
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Sara Siris et al.
(g) Anti-human IgA Alexa Fluor 594: 5 μL.
(h) Anti-human IgD BV 786: 5 μL.
(i) Anti-human IgM BV 650: 5 μL.
(j) Zombie NIR Live/Dead: 1 μL.
3. Incubate the single-stain control tubes on ice for 20 min.
4. For the fluorescence minus one controls, add all antibodies
except the APC antibodies to tube “CF-APC” (see step 1 of
Subheading 3.3.2). Repeat this procedure for other tubes
excluding the relevant antibody for each control. For gating
purposes, add all antibodies to tube “CS.” Incubate all control
tubes on ice for 20 min.
5. Wash all bead and PBMC control tubes with 2 mL ICA buffer.
6. Pellet stained beads and PBMCs at 500 Â g, RT for 8 min.
Discard the supernatant.
7. Resuspend stained beads and PBMCs in 750 μL of ICA buffer.
Transfer these into fresh, labeled 5 mL polystyrene tubes. Store
on ice, in dark, until flow cytometry processing.
After IgG secretion and capture, transfer the sample into a fresh,
labeled (“CS-ICA”) tube and proceed with the following steps:
1. Pellet PBMCs in tube “CS-ICA” at 500 Â g, RT for 8 min.
Discard the supernatant.
2. Resuspend PBMCs in tube “CS-ICA” in 100 μL ICA buffer.
3. Add FAM SE-stained bacteria (at a ratio of 16 bacterial cells to
1 PBMC—see Note 6) to CS-ICA. Incubate at RT, in the dark,
for 20 min.
4. Wash PBMC–bacteria complexes in tube “CS-ICA” with 2 mL
ICA buffer.
5. Pellet PBMC–bacteria complexes at 500 Â g, RT for 8 min.
Discard the supernatant.
6. Resuspend PBMC–bacteria complexes in 74 μL ICA buffer.
7. Prepare a cocktail of all fluorescent antibodies as follows:
(a) Anti-CD3 APC: 2 μL.
(b) Anti-CD14 APC: 1 μL.
(c) Anti-CD19 BV 421: 2 μL.
(d) Anti-CD20 BV 605: 3 μL.
(e) Anti-CD27 PE: 1 μL.
(f) Anti-CD38 PerCP/Cy5.5: 2 μL.
(g) Anti-human IgA Alexa Fluor 594: 5 μL.
(h) Anti-human IgD BV 786: 5 μL.
16
Sara Siris et al.