(b) APC anti-human CD14 antibody (APC).
(c) Brilliant Violet 421™ (BV421) anti-human CD19
antibody.
(d) Brilliant Violet 605™ (BV605) anti-human CD20
antibody.
(e) PE anti-human CD27 antibody (PE).
(f) PerCP/Cyanine5.5 anti-human CD38 antibody (PerCP/
Cy5.5).
(g) Alexa Fluor
® 594 AffiniPure goat anti-human serum IgA,
α chain specific (AF594).
(h) Brilliant Violet 650™ anti-human IgM antibody
(BV650).
(i) Brilliant Violet 785™ anti-human IgD antibody
(BV785).
13. Catch buffer: Nuclease- and protease-free water, 10 mM Tris–
HCL 8.0, 1 U RNAsin.
3 Methods
3.1 Pneumococcal
Growth, Fixing,
and Staining
1. Streak a loopful of pneumococcal frozen stocks on blood agar.
Incubate for 14–16 h at 37
C, 5% CO 2 .
2. From the blood agar plates, suspend 10–20 colonies of pneumococcal growth in 10 mL of BHI-YE culture medium in a
50 mL centrifuge tube.
3. Transfer the pneumococcal suspension to a cell culture flask
with vented cap (see Note 3).
4. Incubate the flask overnight for 14–16 h at 37
C, 5% CO 2
without shaking (see Note 4).
5. Pellet pneumococci from the overnight culture at 3000 Â g for
5 min, RT.
6. Resuspend pneumococcal cells in 10 mL bacterial fixing solution. Incubate for !4 h at RT.
7. Wash pneumococcal cells thrice in 10 mL 0.1 M NaHCO 3 .
Resuspend cells to OD 600 of 0.2 (10
8 cells/mL).
8. Resuspend each aliquot of 10
8 cells in 495 μL of 0.1 M
NaHCO 3 . Add 5 μL of cell staining solution. Incubate for
1 h at 37
C.
9. Wash stained pneumococcal cells five times in 500 μL of 0.1 M
NaHCO 3 .
10. Resuspend stained pneumococcal cells in ICA buffer. Stained
cells can be stored overnight at 4
C, protected from light.
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
13
(c) Brilliant Violet 421™ (BV421) anti-human CD19
antibody.
(d) Brilliant Violet 605™ (BV605) anti-human CD20
antibody.
(e) PE anti-human CD27 antibody (PE).
(f) PerCP/Cyanine5.5 anti-human CD38 antibody (PerCP/
Cy5.5).
(g) Alexa Fluor
® 594 AffiniPure goat anti-human serum IgA,
α chain specific (AF594).
(h) Brilliant Violet 650™ anti-human IgM antibody
(BV650).
(i) Brilliant Violet 785™ anti-human IgD antibody
(BV785).
13. Catch buffer: Nuclease- and protease-free water, 10 mM Tris–
HCL 8.0, 1 U RNAsin.
3 Methods
3.1 Pneumococcal
Growth, Fixing,
and Staining
1. Streak a loopful of pneumococcal frozen stocks on blood agar.
Incubate for 14–16 h at 37
C, 5% CO 2 .
2. From the blood agar plates, suspend 10–20 colonies of pneumococcal growth in 10 mL of BHI-YE culture medium in a
50 mL centrifuge tube.
3. Transfer the pneumococcal suspension to a cell culture flask
with vented cap (see Note 3).
4. Incubate the flask overnight for 14–16 h at 37
C, 5% CO 2
without shaking (see Note 4).
5. Pellet pneumococci from the overnight culture at 3000 Â g for
5 min, RT.
6. Resuspend pneumococcal cells in 10 mL bacterial fixing solution. Incubate for !4 h at RT.
7. Wash pneumococcal cells thrice in 10 mL 0.1 M NaHCO 3 .
Resuspend cells to OD 600 of 0.2 (10
8 cells/mL).
8. Resuspend each aliquot of 10
8 cells in 495 μL of 0.1 M
NaHCO 3 . Add 5 μL of cell staining solution. Incubate for
1 h at 37
C.
9. Wash stained pneumococcal cells five times in 500 μL of 0.1 M
NaHCO 3 .
10. Resuspend stained pneumococcal cells in ICA buffer. Stained
cells can be stored overnight at 4
C, protected from light.
Isolating Bacterial Pathogen-Specific Monoclonal Antibodies
13