6. After 18–20 h, the agarose is removed, and the cells are fixed
with ethanol and stained with Coomassie blue.
7. Count the number of plaques per well and determine the
antibody titer (Table 2).
3.2.4 T Cell Immune
Responses by Detection
of the Stimulated T Cell
Population
1. Humanely euthanize the vaccinated mice using cervical dislocation after anaesthetizing with 100 μl of 80 mg/kg ketamine
and 6 mg/kg xylazine via intraperitoneal injection.
2. Under sterile conditions collect the splenocytes into a 15-ml
centrifuge tube containing Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% heat-inactivated
fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml
streptomycin (DMEM-10).
3. Disrupt the cells by straining through a 100-μm nylon mesh
cell strainer.
4. Remove the red cells with ACK lysis buffer, followed by a
washing step, and transfer the cells to a 50-ml centrifuge tube.
5. Distribute 2 Â 10
6 cells between samples for unstimulated and
stimulated with EFn-3ÂM2e-HA2 plus PA or PA only, phytohemagluttinin, or 10 ng of PMA/100 ng ionomycin (see Note
5).
6. Analyze stimulation of the T cell population after 5 days in the
presence of antigens. Cells are resuspended in BD FACS lysis
buffer, diluted 1:10 in water, and incubated for 10 min at room
temperature. Wash the cells and resuspend in 0.5 ml PBS, then
add 0.5 ml BD Perm2 buffer diluted 1:10 in water, and incubate for 10 min at room temperature. Stain for 30 min with
antibodies targeting T cell markers.
7. Read the samples in an FACSCanto II flow cytometer and
analyze the data with FLOWJO v10 software (FlowJo, Ashland, OR; Table 1).
Table 2
Microneutralization assay: neutralizing antibody titers against PR8 influenza virus
Groups
Titers (2 weeks after the third immunization)
EFn-3ÂM2e-HA2/PA
<10
EFn-3ÂM2e-HA2
<10
PR8
!2560
PBS
<10
The antibodies produce after vaccination with EFn-3ÂM2e-HA2/PA or EFn-3ÂM2e-HA2 could not prevent the
Influenza virus infection to Madin–Darby canine kidney (MDCK) cells. (Adapted from Are ´valo et al. [4])
432
Diana Diaz-Are ´ valo et al.
with ethanol and stained with Coomassie blue.
7. Count the number of plaques per well and determine the
antibody titer (Table 2).
3.2.4 T Cell Immune
Responses by Detection
of the Stimulated T Cell
Population
1. Humanely euthanize the vaccinated mice using cervical dislocation after anaesthetizing with 100 μl of 80 mg/kg ketamine
and 6 mg/kg xylazine via intraperitoneal injection.
2. Under sterile conditions collect the splenocytes into a 15-ml
centrifuge tube containing Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% heat-inactivated
fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml
streptomycin (DMEM-10).
3. Disrupt the cells by straining through a 100-μm nylon mesh
cell strainer.
4. Remove the red cells with ACK lysis buffer, followed by a
washing step, and transfer the cells to a 50-ml centrifuge tube.
5. Distribute 2 Â 10
6 cells between samples for unstimulated and
stimulated with EFn-3ÂM2e-HA2 plus PA or PA only, phytohemagluttinin, or 10 ng of PMA/100 ng ionomycin (see Note
5).
6. Analyze stimulation of the T cell population after 5 days in the
presence of antigens. Cells are resuspended in BD FACS lysis
buffer, diluted 1:10 in water, and incubated for 10 min at room
temperature. Wash the cells and resuspend in 0.5 ml PBS, then
add 0.5 ml BD Perm2 buffer diluted 1:10 in water, and incubate for 10 min at room temperature. Stain for 30 min with
antibodies targeting T cell markers.
7. Read the samples in an FACSCanto II flow cytometer and
analyze the data with FLOWJO v10 software (FlowJo, Ashland, OR; Table 1).
Table 2
Microneutralization assay: neutralizing antibody titers against PR8 influenza virus
Groups
Titers (2 weeks after the third immunization)
EFn-3ÂM2e-HA2/PA
<10
EFn-3ÂM2e-HA2
<10
PR8
!2560
PBS
<10
The antibodies produce after vaccination with EFn-3ÂM2e-HA2/PA or EFn-3ÂM2e-HA2 could not prevent the
Influenza virus infection to Madin–Darby canine kidney (MDCK) cells. (Adapted from Are ´valo et al. [4])
432
Diana Diaz-Are ´ valo et al.