3.4.10 Fixation, Staining,
and Calculation of PRNT 50
Titers
1. After 6 days incubation, add 1.5 mL/well of fixative and
mix well.
2. Leave plates for 1 h at RT.
3. Remove fixative and wash plates with tap water.
4. Add 1.5 mL/well of 1:30 diluted methylene blue dye solution
in water and leave plates for 1 h at RT.
5. Remove dye solution and wash plates with tap water air-dry.
6. Count number of plaques in each well.
7. PRNT 50 titers are given as the reciprocal of serum dilutions
that resulted in a 50% reduction in the number of plaques.
3.5 ELISPOT Assays
1. At the termination of immunogenicity studies, mice are euthanized by cervical dislocation and bones and spleens are collected to enable measurement of antigen-specific memory B
and T cells.
2. Bone marrow (BM) are isolated from femurs by flushing with
3% FCS/PBS in a 2 mL syringe with 25-G needle after cutting
both ends of the bone then pass through 70 μm cell strainer
with a 2 mL syringe plunger.
3. Splenocytes are released by pressing against a 70 μm cell
strainer with a 2 mL syringe plunger.
4. Red blood cells are removed by osmotic shock with RBC lysis
buffer.
5. Cells are washed with 3% FCS/PBS then resuspended in RPMI
complete medium with 10% heat-inactivated FCS.
6. The frequency of antigen-specific antibody- or cytokinesecreting cells is analyzed using biotinylated anti-mouse IgG,
IgG1, IgG2c, or IgM antibodies (Abcam) or anti-mouse
IFN-ɣ, IL-2, IL-4 antibody pairs (BD) or LEAF anti-mouse
IL-17A and biotin-anti-mouse IL-17A antibody (BioLegend)
with streptavidin-HRP (BD Biosciences), according to the
manufacturer’s instruction.
7. Briefly, single-cell suspensions are prepared from BM and
spleens of mice are plated at 2 Â 10
5 cells/well in 96-well
filtration plates pre-coated with antigen of interest (for antibody detection) or anti-mouse cytokine antibodies (for cytokine detection) overnight at 4
C then blocked with RPMI/
10% FCS (see Note 10).
8. For cytokine assays, the cells are incubated with antigen protein
(10 μg/ml) at 37
C and 5% CO 2 for 2 days or 1 day for
antibody producing cell assay (see Note 11).
9. Wells are washed and incubated with biotinylated anti-mouse
cytokine detection antibodies or biotinylated anti-mouse Ig at
RT for 2 h, washed, then streptavidin-HRP added for 1 h
DNA Vaccine Design and Adjuvant Selection
419
and Calculation of PRNT 50
Titers
1. After 6 days incubation, add 1.5 mL/well of fixative and
mix well.
2. Leave plates for 1 h at RT.
3. Remove fixative and wash plates with tap water.
4. Add 1.5 mL/well of 1:30 diluted methylene blue dye solution
in water and leave plates for 1 h at RT.
5. Remove dye solution and wash plates with tap water air-dry.
6. Count number of plaques in each well.
7. PRNT 50 titers are given as the reciprocal of serum dilutions
that resulted in a 50% reduction in the number of plaques.
3.5 ELISPOT Assays
1. At the termination of immunogenicity studies, mice are euthanized by cervical dislocation and bones and spleens are collected to enable measurement of antigen-specific memory B
and T cells.
2. Bone marrow (BM) are isolated from femurs by flushing with
3% FCS/PBS in a 2 mL syringe with 25-G needle after cutting
both ends of the bone then pass through 70 μm cell strainer
with a 2 mL syringe plunger.
3. Splenocytes are released by pressing against a 70 μm cell
strainer with a 2 mL syringe plunger.
4. Red blood cells are removed by osmotic shock with RBC lysis
buffer.
5. Cells are washed with 3% FCS/PBS then resuspended in RPMI
complete medium with 10% heat-inactivated FCS.
6. The frequency of antigen-specific antibody- or cytokinesecreting cells is analyzed using biotinylated anti-mouse IgG,
IgG1, IgG2c, or IgM antibodies (Abcam) or anti-mouse
IFN-ɣ, IL-2, IL-4 antibody pairs (BD) or LEAF anti-mouse
IL-17A and biotin-anti-mouse IL-17A antibody (BioLegend)
with streptavidin-HRP (BD Biosciences), according to the
manufacturer’s instruction.
7. Briefly, single-cell suspensions are prepared from BM and
spleens of mice are plated at 2 Â 10
5 cells/well in 96-well
filtration plates pre-coated with antigen of interest (for antibody detection) or anti-mouse cytokine antibodies (for cytokine detection) overnight at 4
C then blocked with RPMI/
10% FCS (see Note 10).
8. For cytokine assays, the cells are incubated with antigen protein
(10 μg/ml) at 37
C and 5% CO 2 for 2 days or 1 day for
antibody producing cell assay (see Note 11).
9. Wells are washed and incubated with biotinylated anti-mouse
cytokine detection antibodies or biotinylated anti-mouse Ig at
RT for 2 h, washed, then streptavidin-HRP added for 1 h
DNA Vaccine Design and Adjuvant Selection
419