15. Prepare gradients immediately before use as preparing the
gradient in advance will result in diffusion between the two
layers and suboptimal neutrophil purity and recovery.
16. Overlaying the bone marrow cell suspension over Histopaque
1077 needs to be done slowly in order to avoid disturbing the
interface between the cells and Histopaque 1077. To do so,
dispense drop by drop in order to avoid mixing the two densities, which will preclude cell separation during centrifugation.
17. Centrifugation of the gradient at room temperature is critical
and essential for effective separation of the neutrophils.
18. Neutrophils are typically >95% viable and >90% pure as determined by FACS analysis. The typical yield of neutrophils from
the bone marrow (i.e., two femur and two tibia bones) of an
uninfected 8- to 12-week-old C57BL/6 mouse is ~4–6 million
cells. In a situation when two distinct layers of cells are not
seen, the researcher is advised to collect the entire cell layer in a
new 15 ml conical tube and wash the cells once in RPMI + 10%
FBS and resuspend in 1.0 ml of PBS and repeat the
Histopaque step.
19. We usually resuspend the PMNs in ÀÀ+ media (HBSS Ca/
Mg-free with 0.1% gelatin) as the glucose in the HBSSÀ maintains PMN viability while the gelatin prevents cell clumping
and the buffer is Ca/Mg-free to prevent activation.
20. For OPH assay, prepare fresh 2% gelatin and all the buffers on
the day of experiment. Any possible contamination in previously prepared buffers could prematurely activate PMNs.
21. This assay can be technically challenging as PMNs can be
activated easily which can lead to variability. We recommend
to test each condition in at least quadruplicates, particularly for
beginners.
22. Avoid using any tissue culture treated plastic, as this may cause
PMNs to adhere and get activated.
23. A standard number of opsonized bacteria are added to the
reaction mix containing PMNs and the survival percentage is
calculated at the end of the reaction to estimate the killing
efficiency. In these assays, we typically aim for 10
3
S. pneumoniae
per 10
5 PMNs and 3% sera in a 100 μl reaction. These conditions have been optimized for the following S. pneumoniae
strains: TIGR4, 19F, 23F, and D39.
24. S. pneumoniae aliquots frozen down at midlogarithmic phase
or grown the day of the assay till mid-logarithmic phase can
both be used for this assay.
572
Manmeet Bhalla et al.
gradient in advance will result in diffusion between the two
layers and suboptimal neutrophil purity and recovery.
16. Overlaying the bone marrow cell suspension over Histopaque
1077 needs to be done slowly in order to avoid disturbing the
interface between the cells and Histopaque 1077. To do so,
dispense drop by drop in order to avoid mixing the two densities, which will preclude cell separation during centrifugation.
17. Centrifugation of the gradient at room temperature is critical
and essential for effective separation of the neutrophils.
18. Neutrophils are typically >95% viable and >90% pure as determined by FACS analysis. The typical yield of neutrophils from
the bone marrow (i.e., two femur and two tibia bones) of an
uninfected 8- to 12-week-old C57BL/6 mouse is ~4–6 million
cells. In a situation when two distinct layers of cells are not
seen, the researcher is advised to collect the entire cell layer in a
new 15 ml conical tube and wash the cells once in RPMI + 10%
FBS and resuspend in 1.0 ml of PBS and repeat the
Histopaque step.
19. We usually resuspend the PMNs in ÀÀ+ media (HBSS Ca/
Mg-free with 0.1% gelatin) as the glucose in the HBSSÀ maintains PMN viability while the gelatin prevents cell clumping
and the buffer is Ca/Mg-free to prevent activation.
20. For OPH assay, prepare fresh 2% gelatin and all the buffers on
the day of experiment. Any possible contamination in previously prepared buffers could prematurely activate PMNs.
21. This assay can be technically challenging as PMNs can be
activated easily which can lead to variability. We recommend
to test each condition in at least quadruplicates, particularly for
beginners.
22. Avoid using any tissue culture treated plastic, as this may cause
PMNs to adhere and get activated.
23. A standard number of opsonized bacteria are added to the
reaction mix containing PMNs and the survival percentage is
calculated at the end of the reaction to estimate the killing
efficiency. In these assays, we typically aim for 10
3
S. pneumoniae
per 10
5 PMNs and 3% sera in a 100 μl reaction. These conditions have been optimized for the following S. pneumoniae
strains: TIGR4, 19F, 23F, and D39.
24. S. pneumoniae aliquots frozen down at midlogarithmic phase
or grown the day of the assay till mid-logarithmic phase can
both be used for this assay.
572
Manmeet Bhalla et al.