(g) “CF-PerCP/Cy5.5” for fluorescence minus PerCP/
Cy5.5 control.
(h) “CF-AF594” for fluorescence minus AF594 control.
(i) “CF-BV785” for fluorescence minus BV785 control.
(j) “CF-BV650” for fluorescence minus BV650 control.
(k) “CF-Zombie” for fluorescence minus Zombie NIR
control.
(l) “CS-ICA” for stained PBMCs (ICA).
2. Transfer 100 μL of PBMCs to each tube. Store tubes (a)–(k)
above on ice (see Note 5).
3. Add 1 μL of Zombie NIR live/dead dye to PBMCs in tube
“CS-ICA”. Incubate at RT for 20 min, in dark.
4. Wash PBMCs in tube “CS-ICA” with 2 mL ICA buffer.
5. Pellet PBMCs in tube “CS-ICA” at 500 Â g, RT for 8 min.
Discard the supernatant.
6. Resuspend PBMCs in tube “CS-ICA” in 100 μL ICA buffer.
7. Add 1 μg of the anti-human CD45 Streptavidin conjugate to
PBMCs in tube “CS-ICA.” Mix contents of the tube by gentle
tapping. Incubate on ice for 20 min.
8. Repeat steps 4–6.
9. Add 1 μL of the Biotin anti-human IgG antibody PBMCs in
tube “CS-ICA.” Mix contents of the tube by gentle tapping.
Incubate on ice for 20 min.
10. Repeat steps 4 and 5.
11. Resuspend PBMCs in tube “CS-ICA” in 1 mL of R10. Transfer contents of this tube to a fresh 2 mL microcentrifuge tube.
Label the new tube as “CS-ICA.”
12. For secretion and capture of IgG, cap the tube tightly and
incubate at 37
C for 1 h on a tube rotator.
During the 1 h secretion/capture step, prepare the single stain and
fluorescence minus one controls:
1. Vortex the single-color compensation beads. Transfer 1 drop
each (~50 μL) into separate labeled tubes, one tube for each
fluorescent antibody.
2. Add each antibody to its corresponding tube, 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.
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
15
Cy5.5 control.
(h) “CF-AF594” for fluorescence minus AF594 control.
(i) “CF-BV785” for fluorescence minus BV785 control.
(j) “CF-BV650” for fluorescence minus BV650 control.
(k) “CF-Zombie” for fluorescence minus Zombie NIR
control.
(l) “CS-ICA” for stained PBMCs (ICA).
2. Transfer 100 μL of PBMCs to each tube. Store tubes (a)–(k)
above on ice (see Note 5).
3. Add 1 μL of Zombie NIR live/dead dye to PBMCs in tube
“CS-ICA”. Incubate at RT for 20 min, in dark.
4. Wash PBMCs in tube “CS-ICA” with 2 mL ICA buffer.
5. Pellet PBMCs in tube “CS-ICA” at 500 Â g, RT for 8 min.
Discard the supernatant.
6. Resuspend PBMCs in tube “CS-ICA” in 100 μL ICA buffer.
7. Add 1 μg of the anti-human CD45 Streptavidin conjugate to
PBMCs in tube “CS-ICA.” Mix contents of the tube by gentle
tapping. Incubate on ice for 20 min.
8. Repeat steps 4–6.
9. Add 1 μL of the Biotin anti-human IgG antibody PBMCs in
tube “CS-ICA.” Mix contents of the tube by gentle tapping.
Incubate on ice for 20 min.
10. Repeat steps 4 and 5.
11. Resuspend PBMCs in tube “CS-ICA” in 1 mL of R10. Transfer contents of this tube to a fresh 2 mL microcentrifuge tube.
Label the new tube as “CS-ICA.”
12. For secretion and capture of IgG, cap the tube tightly and
incubate at 37
C for 1 h on a tube rotator.
During the 1 h secretion/capture step, prepare the single stain and
fluorescence minus one controls:
1. Vortex the single-color compensation beads. Transfer 1 drop
each (~50 μL) into separate labeled tubes, one tube for each
fluorescent antibody.
2. Add each antibody to its corresponding tube, 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.
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
15