it is possible to use cell-permeable amine-binding dyes. Bacterial cells are considerably smaller than mammalian cells and
therefore the side scatter and forward scatter on the flow cytometer have to be adjusted accordingly. Operation of flow
cytometers is beyond the scope of this chapter.
9. This procedure is adapted from a method originally published
by Moor et al. [18] and tailored to quantify the amount of
antibodies bound on the surface of S. pneumoniae TIGR4
strain opsonized with different sera. We recommend using
1%, 3% or 10% of immune-sera (of the total reaction volume;
i.e., 0.5, 1.5, or 5 μl correspondingly per well). These dilutions
provide a dose-dependent response and can be used to parse
out differences between immune and naı ¨ve sera (Fig. 2a, b). No
increase in the amounts of surface-bound antibodies is seen
above 10% using immune sera indicating the signal is saturated.
Sera from naı ¨ve-unimmunized mice should be included as a
control for the baseline amounts of cross-reactive antibodies
found in the sera. Hyperimmune sera (Subheading 3.3) can be
used as an internal control for interassay variation.
10. The use of necessary controls to exclude the possibility of
nonspecific binding is advised. To ensure there is no crossreactivity between the secondary antibody and bacterial surface, stain unopsonized bacteria (bacteria not incubated with
any sera) with the secondary antibody alone. To determine the
specificity of the Prevnar 13
® vaccine response, we also used an
S. pneumoniae capsule deletion mutant (Δcps) and detected no
fluorescent signal compared to the wild-type TIGR4 strain
indicating the specificity of the antibodies against surface polysaccharide (Fig. 2c).
11. To isolate bone-marrow derived PMNs we use a Histopaque
density based centrifugation method using a protocol established by Swamydas and Lionakis [16] with very few modifications. Make sure to cite the Swamydas and Lionakis reference
in publications using the Histopaque PMN isolation protocol.
Other density based centrifugation methods using Percoll [19]
or negative/positive selection methods using commercial kits
may also be used.
12. Blanching of bones indicates that the bone marrow cells have
been flushed out. Use approximately 10 ml of media to flush a
set of femur–tibia pairs. Adding EDTA to the medium is
important to prevent cell clumping.
13. Following isolation of cells from bone marrow, avoid using any
buffer or media containing calcium (Ca
++ ). This is to avoid
premature neutrophil activation prior to bacterial challenge.
14. Histopaque 1119 and Histopaque 1077 should be warmed to
18–26
C before use.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
571
therefore the side scatter and forward scatter on the flow cytometer have to be adjusted accordingly. Operation of flow
cytometers is beyond the scope of this chapter.
9. This procedure is adapted from a method originally published
by Moor et al. [18] and tailored to quantify the amount of
antibodies bound on the surface of S. pneumoniae TIGR4
strain opsonized with different sera. We recommend using
1%, 3% or 10% of immune-sera (of the total reaction volume;
i.e., 0.5, 1.5, or 5 μl correspondingly per well). These dilutions
provide a dose-dependent response and can be used to parse
out differences between immune and naı ¨ve sera (Fig. 2a, b). No
increase in the amounts of surface-bound antibodies is seen
above 10% using immune sera indicating the signal is saturated.
Sera from naı ¨ve-unimmunized mice should be included as a
control for the baseline amounts of cross-reactive antibodies
found in the sera. Hyperimmune sera (Subheading 3.3) can be
used as an internal control for interassay variation.
10. The use of necessary controls to exclude the possibility of
nonspecific binding is advised. To ensure there is no crossreactivity between the secondary antibody and bacterial surface, stain unopsonized bacteria (bacteria not incubated with
any sera) with the secondary antibody alone. To determine the
specificity of the Prevnar 13
® vaccine response, we also used an
S. pneumoniae capsule deletion mutant (Δcps) and detected no
fluorescent signal compared to the wild-type TIGR4 strain
indicating the specificity of the antibodies against surface polysaccharide (Fig. 2c).
11. To isolate bone-marrow derived PMNs we use a Histopaque
density based centrifugation method using a protocol established by Swamydas and Lionakis [16] with very few modifications. Make sure to cite the Swamydas and Lionakis reference
in publications using the Histopaque PMN isolation protocol.
Other density based centrifugation methods using Percoll [19]
or negative/positive selection methods using commercial kits
may also be used.
12. Blanching of bones indicates that the bone marrow cells have
been flushed out. Use approximately 10 ml of media to flush a
set of femur–tibia pairs. Adding EDTA to the medium is
important to prevent cell clumping.
13. Following isolation of cells from bone marrow, avoid using any
buffer or media containing calcium (Ca
++ ). This is to avoid
premature neutrophil activation prior to bacterial challenge.
14. Histopaque 1119 and Histopaque 1077 should be warmed to
18–26
C before use.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
571