16. Discard the supernatant and resuspend the pellet in 1 ml of
sterile PBS (room temperature).
17. In a sterile 15 ml conical tube, add 3 ml of Histopaque 1119
(density, 1.119 g/ml). Very slowly overlay 3 ml of Histopaque
1077 (density, 1.077 g/ml) on the 3 ml of Histopaque 1119
(see Notes 14–16).
18. Slowly overlay the bone marrow cells (from step 16) on top of
the Histopaque 1077.
19. Centrifuge for 30 min at 913 rcf at 25
C without brake (OFF)
(see Note 17).
20. Collect the PMNs at the interface of the Histopaque 1119 and
Histopaque 1077 layers. Collect the lower layer only as it is the
one containing mature PMNs (Ly6G
hi ). Make sure to place
your pipette tip on top of the layer and as you draw the liquid
up, rotate your pipette to collect all the cells. Transfer the cells
into a new 15-ml conical tube (see Note 18).
21. Add 5 ml RP-10 and centrifuge at 328 rcf for 5 min at 4
C.
22. Remove supernatant and resuspend the cell pellet with 5 mL
RP-10. At this point, take 10 μl out and mix with 10 μl of
trypan blue for counting the cell number.
23. Centrifuge the remaining volume at 328 rcf for 5 min at 4
C.
During, count the neutrophils using a Hemocytometer
and determine their viability by trypan blue exclusion. Bright
cells are alive, while cells that appear blue are cells that have lost
their membrane integrity. Count under the microscope (the
4 Â 5 squares filled with small squares surrounded by double
lines).
Cell numbers: Average count  2 (df)  10
4
 5 (volume
of cell suspension in step 22).
– df is the dilution factor (10 μl of the cell suspension + 10 μl
of Trypan blue dye).
– 10
4 is to convert the cell count to cells per ml as each square
has volume of 10
À4 ml.
24. Resuspend the cells in ÀÀ+ buffer at the desired cell number.
Keep the cells on ice until use. At this point, PMNs can stay on
ice for 2–3 h without significant effect on their viability (see
Note 19). Cell purity can be determined by staining with antiLy6G and CD11b antibodies [10, 16].
3.7 Opsonophagocytic Killing
Assay (OPH) Using
Primary Neutrophils
1. The day of the assay prepare +++ buffer (Hanks (HBSS+) with
calcium and magnesium and 0.1% gelatin (see Note 20). We
recommend testing each condition at least in quadruplicates
(see Note 21).
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
567
sterile PBS (room temperature).
17. In a sterile 15 ml conical tube, add 3 ml of Histopaque 1119
(density, 1.119 g/ml). Very slowly overlay 3 ml of Histopaque
1077 (density, 1.077 g/ml) on the 3 ml of Histopaque 1119
(see Notes 14–16).
18. Slowly overlay the bone marrow cells (from step 16) on top of
the Histopaque 1077.
19. Centrifuge for 30 min at 913 rcf at 25
C without brake (OFF)
(see Note 17).
20. Collect the PMNs at the interface of the Histopaque 1119 and
Histopaque 1077 layers. Collect the lower layer only as it is the
one containing mature PMNs (Ly6G
hi ). Make sure to place
your pipette tip on top of the layer and as you draw the liquid
up, rotate your pipette to collect all the cells. Transfer the cells
into a new 15-ml conical tube (see Note 18).
21. Add 5 ml RP-10 and centrifuge at 328 rcf for 5 min at 4
C.
22. Remove supernatant and resuspend the cell pellet with 5 mL
RP-10. At this point, take 10 μl out and mix with 10 μl of
trypan blue for counting the cell number.
23. Centrifuge the remaining volume at 328 rcf for 5 min at 4
C.
During, count the neutrophils using a Hemocytometer
and determine their viability by trypan blue exclusion. Bright
cells are alive, while cells that appear blue are cells that have lost
their membrane integrity. Count under the microscope (the
4 Â 5 squares filled with small squares surrounded by double
lines).
Cell numbers: Average count  2 (df)  10
4
 5 (volume
of cell suspension in step 22).
– df is the dilution factor (10 μl of the cell suspension + 10 μl
of Trypan blue dye).
– 10
4 is to convert the cell count to cells per ml as each square
has volume of 10
À4 ml.
24. Resuspend the cells in ÀÀ+ buffer at the desired cell number.
Keep the cells on ice until use. At this point, PMNs can stay on
ice for 2–3 h without significant effect on their viability (see
Note 19). Cell purity can be determined by staining with antiLy6G and CD11b antibodies [10, 16].
3.7 Opsonophagocytic Killing
Assay (OPH) Using
Primary Neutrophils
1. The day of the assay prepare +++ buffer (Hanks (HBSS+) with
calcium and magnesium and 0.1% gelatin (see Note 20). We
recommend testing each condition at least in quadruplicates
(see Note 21).
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
567