(i) Anti-human IgM BV 650: 5 μL.
(j) Add the cocktail of antibodies to tube CS-ICA (see Note
7).
8. Incubate the “CS-ICA” tube on ice for 20 min, in the dark.
9. Wash stained PBMC–bacteria complexes with 2 mL ICA
buffer.
10. Pellet stained PBMC–bacteria complexes at 500 Â g, RT for
8 min. Discard the supernatant.
11. Resuspend PBMC–bacteria complexes in 750 μL of ICA
buffer. Transfer these into a fresh, labeled 5 mL
polystyrene tube.
12. Pass PBMC controls and experiment samples through strainers
into fresh, labeled polystyrene tubes to avoid clogging the
sorter.
13. Gate for live, pathogen-specific plasmablasts as follows:
(a) CD3 negative, CD14 negative.
(b) CD19 positive, CD 20 low/negative.
(c) CD27 high, CD38 high.
(d) IgD negative, IgA negative.
(e) IgM negative, FAM SE positive.
14. Sort pathogen-specific plasmablasts singly into rows A–G of a
96-well plate containing 10 μL of catch buffer. Row H will
serve as negative control in downstream PCRs.
15. Refer to previously published protocols for VH/VL amplification and cloning from individual plasmablasts and hmAb
expression [2, 6].
4 Notes
1. It is expected that some cell lysis will occur during PBMC
thawing and/or during the assay. Released nucleic acid from
lysed cells leads to cell clumping, which significantly reduces
recovery of single cells available for FACS. We found that
including Benzonase Nuclease (Fisher Scientific, #10725899)
in the assay buffer prevented cell clumping, ensured easy resuspension of cells following the wash steps and allowed for full
PBMC recovery at the end of the assay.
2. We used the Lightning-Link
® streptavidin antibody labeling kit
(Expedeon, #708-0030) and an anti-human CD45 antibody
(BioLegend, #304002).
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
17
(j) Add the cocktail of antibodies to tube CS-ICA (see Note
7).
8. Incubate the “CS-ICA” tube on ice for 20 min, in the dark.
9. Wash stained PBMC–bacteria complexes with 2 mL ICA
buffer.
10. Pellet stained PBMC–bacteria complexes at 500 Â g, RT for
8 min. Discard the supernatant.
11. Resuspend PBMC–bacteria complexes in 750 μL of ICA
buffer. Transfer these into a fresh, labeled 5 mL
polystyrene tube.
12. Pass PBMC controls and experiment samples through strainers
into fresh, labeled polystyrene tubes to avoid clogging the
sorter.
13. Gate for live, pathogen-specific plasmablasts as follows:
(a) CD3 negative, CD14 negative.
(b) CD19 positive, CD 20 low/negative.
(c) CD27 high, CD38 high.
(d) IgD negative, IgA negative.
(e) IgM negative, FAM SE positive.
14. Sort pathogen-specific plasmablasts singly into rows A–G of a
96-well plate containing 10 μL of catch buffer. Row H will
serve as negative control in downstream PCRs.
15. Refer to previously published protocols for VH/VL amplification and cloning from individual plasmablasts and hmAb
expression [2, 6].
4 Notes
1. It is expected that some cell lysis will occur during PBMC
thawing and/or during the assay. Released nucleic acid from
lysed cells leads to cell clumping, which significantly reduces
recovery of single cells available for FACS. We found that
including Benzonase Nuclease (Fisher Scientific, #10725899)
in the assay buffer prevented cell clumping, ensured easy resuspension of cells following the wash steps and allowed for full
PBMC recovery at the end of the assay.
2. We used the Lightning-Link
® streptavidin antibody labeling kit
(Expedeon, #708-0030) and an anti-human CD45 antibody
(BioLegend, #304002).
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
17