m. injections. Gently pull out the leg to be injected through the
restrainer and locate the caudal thigh muscle. While holding
the foot firmly, swab the site with 70% ethanol, aspirate and
inject the bacterial suspension with the needle bevel up.
3. The same protocol can be used for immunizing mice with
Pneumovax
® . S. pneumoniae TIGR4 used here, is a serotype
4 strain covered by both vaccines.
4. For intranasal inoculation, restrain the mouse without anesthesia in a way and position similar to during intraperitoneal (i.p.)
injections. Make sure their head is held steady and wait for
normal breathing. Pipet 10 μl of inoculum. Make a drop
(with approximately half the volume of the inoculum) on the
tip of pipette and deliver into a nostril. Do not touch the nose
with tip of pipette since it will induce sneezing. Approach the
mouse from the front, opposite to the eyes so that the mouse
does not see you (otherwise the mice will try to eat the drop).
Deliver the rest to the other nostril the same way. Alternatively,
the whole inoculum can be delivered in a single drop between
the two nostrils.
5. For timing consideration, hyperimmune sera is achieved by
intranasally vaccinating the mice with live bacteria (once weekly
for 3 consecutive weeks), followed by immunizing with Prevnar 13
® . The procedure requires 4 weeks for the successive
treatments followed by 4 weeks until sera collection and produces very high titers of anti-pneumococcal IgG and IgM
[17]. If higher antibody titers are desired, heat-killed bacteria
can also be injected (i.p.) 4 weeks following Prevnar 13
® and
mice can be bled a week after the injection. We typically prepare
hyperimmune sera once a year using 40 mice which yields
28 ml of sera. The antibody titer in the hyperimmune sera
can be determined by ELISA as previously described [17].
6. While euthanizing mice for immune sera, it is advised to handle
one animal at a time. Once the blood has been transferred to
Microtainer tube, proceed with the next mouse. Euthanizing
multiple mice at one time could cause delay and coagulation of
blood. Do not allow the blood or the sera to sit for a long time
in the Microtainer tubes. Once spun, immediately transfer to a
15-ml conical tube or a 1.5 ml microfuge tube.
7. Make sure the sera are saved at À80
C. We have noticed that if
the sera are saved at higher temperatures (e.g., À20
C), then
with time they become toxic to the bacteria and their ability to
induce opsonophagocytic killing decreases.
8. This assay can be used with S. pneumoniae strain not expressing
GFP (or other fluorescent) tags. However, fluorescent tagging
can help with gating on the bacteria during flow cytometry
analysis. For pneumococcal strains lacking fluorescent proteins,
570
Manmeet Bhalla et al.
restrainer and locate the caudal thigh muscle. While holding
the foot firmly, swab the site with 70% ethanol, aspirate and
inject the bacterial suspension with the needle bevel up.
3. The same protocol can be used for immunizing mice with
Pneumovax
® . S. pneumoniae TIGR4 used here, is a serotype
4 strain covered by both vaccines.
4. For intranasal inoculation, restrain the mouse without anesthesia in a way and position similar to during intraperitoneal (i.p.)
injections. Make sure their head is held steady and wait for
normal breathing. Pipet 10 μl of inoculum. Make a drop
(with approximately half the volume of the inoculum) on the
tip of pipette and deliver into a nostril. Do not touch the nose
with tip of pipette since it will induce sneezing. Approach the
mouse from the front, opposite to the eyes so that the mouse
does not see you (otherwise the mice will try to eat the drop).
Deliver the rest to the other nostril the same way. Alternatively,
the whole inoculum can be delivered in a single drop between
the two nostrils.
5. For timing consideration, hyperimmune sera is achieved by
intranasally vaccinating the mice with live bacteria (once weekly
for 3 consecutive weeks), followed by immunizing with Prevnar 13
® . The procedure requires 4 weeks for the successive
treatments followed by 4 weeks until sera collection and produces very high titers of anti-pneumococcal IgG and IgM
[17]. If higher antibody titers are desired, heat-killed bacteria
can also be injected (i.p.) 4 weeks following Prevnar 13
® and
mice can be bled a week after the injection. We typically prepare
hyperimmune sera once a year using 40 mice which yields
28 ml of sera. The antibody titer in the hyperimmune sera
can be determined by ELISA as previously described [17].
6. While euthanizing mice for immune sera, it is advised to handle
one animal at a time. Once the blood has been transferred to
Microtainer tube, proceed with the next mouse. Euthanizing
multiple mice at one time could cause delay and coagulation of
blood. Do not allow the blood or the sera to sit for a long time
in the Microtainer tubes. Once spun, immediately transfer to a
15-ml conical tube or a 1.5 ml microfuge tube.
7. Make sure the sera are saved at À80
C. We have noticed that if
the sera are saved at higher temperatures (e.g., À20
C), then
with time they become toxic to the bacteria and their ability to
induce opsonophagocytic killing decreases.
8. This assay can be used with S. pneumoniae strain not expressing
GFP (or other fluorescent) tags. However, fluorescent tagging
can help with gating on the bacteria during flow cytometry
analysis. For pneumococcal strains lacking fluorescent proteins,
570
Manmeet Bhalla et al.