3.2.5 Tetramers
and Staining
1. The following tetramers were obtained through the NIH Tetramer Core Facility at Emory University (Atlanta, GA): The
MHC Class II tetramer I-A(d) / SLLTEVETPIRNEWGS and
MHC Class II tetramer H-2K(d)/VETPIRNEW (influenza
epitope) are labeled with phycoerythrin and allophycocyanin.
2. Stain 1 Â 10
6 splenic mononuclear cells/ml with 1 μg MHC
class II tetramer and incubate for 3 h at 37
. Wash the collected
samples by centrifugation (using staining buffer at 800 Â g for
5 min).
3. Stain the cells with anti-mouse CD4–allophycocyanin and
CD3–Alexa Fluor 700 for 30 min at room temperature in
the dark.
4. For MHC class I experiments, label the cells with a 1:100
dilution of tetramer for 30 min at room temperature. After
washing, stain with anti-mouse CD8–phycoerythrin and CD3–
FITC (BD Biosciences) for 30 min at room temperature in
the dark.
5. Wash the samples with staining buffer at 800 Â g for 5 min and
resuspend in 300 μl of 2% formaldehyde in PBS.
6. Read the samples in an FACSCanto II flow cytometer and
analyze the data with FLOWJO v10 software (FlowJo,
Ashland, OR).
3.2.6 Lethal Challenge
with Influenza Viruses
1. Challenge the vaccinated mice with H1N1 influenza virus
PR8 2 weeks after administration of the third vaccine dose
(see Note 2).
2. Weigh each mouse and then again after being anaesthetized
with ketamine and xylazine.
3. Challenge the mice intranasally with 2 Â 10
5 PFU of live
A/PR/8/34 virus in 40 μl.
4. Challenge the mice intranasally with heterologous
1 Â 10
5 PFU of live A/CA/4/09 virus in 40 μl.
5. Monitor the mice for signs of illness and weigh daily for 21 days
postinoculation.
6. Death or 30% weight loss is considered an endpoint for survival
analysis.
7. The results are showing in the Fig. 3. Survival of vaccinated
mice following lethal challenge with influenza A. PR8 (H1N1)
and B with CA09 virus.
3.2.7 Lethal Challenge
with B. anthracis Sterne
Spores
1. A/J (6–8 week) mice are sensitive to receiving an infection with
B. anthracis Sterne spores (used to vaccinate military personal).
These mice lack complement, making them susceptible to
lethal challenge.
Vaccine Delivery with a Detoxified Bacterial Toxin
433
and Staining
1. The following tetramers were obtained through the NIH Tetramer Core Facility at Emory University (Atlanta, GA): The
MHC Class II tetramer I-A(d) / SLLTEVETPIRNEWGS and
MHC Class II tetramer H-2K(d)/VETPIRNEW (influenza
epitope) are labeled with phycoerythrin and allophycocyanin.
2. Stain 1 Â 10
6 splenic mononuclear cells/ml with 1 μg MHC
class II tetramer and incubate for 3 h at 37
. Wash the collected
samples by centrifugation (using staining buffer at 800 Â g for
5 min).
3. Stain the cells with anti-mouse CD4–allophycocyanin and
CD3–Alexa Fluor 700 for 30 min at room temperature in
the dark.
4. For MHC class I experiments, label the cells with a 1:100
dilution of tetramer for 30 min at room temperature. After
washing, stain with anti-mouse CD8–phycoerythrin and CD3–
FITC (BD Biosciences) for 30 min at room temperature in
the dark.
5. Wash the samples with staining buffer at 800 Â g for 5 min and
resuspend in 300 μl of 2% formaldehyde in PBS.
6. Read the samples in an FACSCanto II flow cytometer and
analyze the data with FLOWJO v10 software (FlowJo,
Ashland, OR).
3.2.6 Lethal Challenge
with Influenza Viruses
1. Challenge the vaccinated mice with H1N1 influenza virus
PR8 2 weeks after administration of the third vaccine dose
(see Note 2).
2. Weigh each mouse and then again after being anaesthetized
with ketamine and xylazine.
3. Challenge the mice intranasally with 2 Â 10
5 PFU of live
A/PR/8/34 virus in 40 μl.
4. Challenge the mice intranasally with heterologous
1 Â 10
5 PFU of live A/CA/4/09 virus in 40 μl.
5. Monitor the mice for signs of illness and weigh daily for 21 days
postinoculation.
6. Death or 30% weight loss is considered an endpoint for survival
analysis.
7. The results are showing in the Fig. 3. Survival of vaccinated
mice following lethal challenge with influenza A. PR8 (H1N1)
and B with CA09 virus.
3.2.7 Lethal Challenge
with B. anthracis Sterne
Spores
1. A/J (6–8 week) mice are sensitive to receiving an infection with
B. anthracis Sterne spores (used to vaccinate military personal).
These mice lack complement, making them susceptible to
lethal challenge.
Vaccine Delivery with a Detoxified Bacterial Toxin
433