2. Label the reaction plate (non–tissue culture treated round
bottom 96-well plate) (see Note 22) and add +++ buffer as
follows:
(a) Add 50 μl of +++ buffer to ALL wells.
(b) Add 40 μl of +++ buffer to No PMN wells.
(c) Leave at room temp until PMNs are ready to be added.
The abovementioned volumes can be adjusted as needed to
scale up or down. It is crucial to keep the final volumes the same
in +PMNs and No PMNs wells and across different groups.
3. Dilute mid-logarithmic phase grown S. pneumoniae to 10
5
/ml
in +++ buffer (see Notes 23–25).
4. For opsonization of S. pneumoniae, mix bacteria with the target
mouse sera. To achieve 3% sera in the final reaction, mix 3 μl
sera with 10 μl (10
3 ) bacteria. A master mix should be prepared
to use in replicate wells taking into account +PMN and No
PMN controls. Always prepare for three extra wells. For example, if you have 12 experimental wells total, prepare for 15 wells
by mixing 45 μl sera with 150 μl of diluted bacteria in a
microcentrifuge tube.
5. Incubate the reactions at 37
C for 30 min in a shaking incubator with the speed set at 78 rpm (see Note 26). The ratio of
bacteria to immune sera in the final reaction should be adjusted
as needed (see Note 27).
6. During the last 5 min of above incubation, add 10
5 PMNs
(40 μl) to the “PMN wells” of your reaction plate containing +
++ buffer. Leave the plate at room temperature till the bacteria
are ready to be added.
7. Once S. pneumoniae are opsonized, add 13 μl of bacteria/sera
mix to all the wells (PMNs and No PMNs). Use of multichannel pipette is advised for speed and to avoid technical error in
dispensing equal number of bacteria.
8. Incubate the plate at 37
C shaking (78 rpm) for 40 min.
During this incubation, titer the bacteria/sera mix by plating
serial dilutions on blood agar plates to estimate the bacterial
“Input.”
9. Once the 40 min incubation is over (step 8), place the reaction
plate on ice for ~2 min to stop the reaction. Immediately
afterward, plate 10 μl volumes of the reaction mix on blood
agar plates using the drip method. Plating the zero dilution
(directly from the plate) should allow for countable colonies.
The reaction mix can also be diluted and then plated if more
than 10
3 bacteria per reaction are being used (see Note 28).
10. Incubate the plates overnight at 37
C + 5% CO 2 . Next day,
count the number of colonies (see Notes 29–31) (Fig. 3).
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Manmeet Bhalla et al.
bottom 96-well plate) (see Note 22) and add +++ buffer as
follows:
(a) Add 50 μl of +++ buffer to ALL wells.
(b) Add 40 μl of +++ buffer to No PMN wells.
(c) Leave at room temp until PMNs are ready to be added.
The abovementioned volumes can be adjusted as needed to
scale up or down. It is crucial to keep the final volumes the same
in +PMNs and No PMNs wells and across different groups.
3. Dilute mid-logarithmic phase grown S. pneumoniae to 10
5
/ml
in +++ buffer (see Notes 23–25).
4. For opsonization of S. pneumoniae, mix bacteria with the target
mouse sera. To achieve 3% sera in the final reaction, mix 3 μl
sera with 10 μl (10
3 ) bacteria. A master mix should be prepared
to use in replicate wells taking into account +PMN and No
PMN controls. Always prepare for three extra wells. For example, if you have 12 experimental wells total, prepare for 15 wells
by mixing 45 μl sera with 150 μl of diluted bacteria in a
microcentrifuge tube.
5. Incubate the reactions at 37
C for 30 min in a shaking incubator with the speed set at 78 rpm (see Note 26). The ratio of
bacteria to immune sera in the final reaction should be adjusted
as needed (see Note 27).
6. During the last 5 min of above incubation, add 10
5 PMNs
(40 μl) to the “PMN wells” of your reaction plate containing +
++ buffer. Leave the plate at room temperature till the bacteria
are ready to be added.
7. Once S. pneumoniae are opsonized, add 13 μl of bacteria/sera
mix to all the wells (PMNs and No PMNs). Use of multichannel pipette is advised for speed and to avoid technical error in
dispensing equal number of bacteria.
8. Incubate the plate at 37
C shaking (78 rpm) for 40 min.
During this incubation, titer the bacteria/sera mix by plating
serial dilutions on blood agar plates to estimate the bacterial
“Input.”
9. Once the 40 min incubation is over (step 8), place the reaction
plate on ice for ~2 min to stop the reaction. Immediately
afterward, plate 10 μl volumes of the reaction mix on blood
agar plates using the drip method. Plating the zero dilution
(directly from the plate) should allow for countable colonies.
The reaction mix can also be diluted and then plated if more
than 10
3 bacteria per reaction are being used (see Note 28).
10. Incubate the plates overnight at 37
C + 5% CO 2 . Next day,
count the number of colonies (see Notes 29–31) (Fig. 3).
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Manmeet Bhalla et al.