3. The blocking buffer is replaced with a primary antibody in the
same buffer for 30 min at room temperature and washed three
times with PBS and incubated with a secondary antibody for
30 min at room temperature.
4. Washed coverslips are rewashed three times with PBS, once
more with H 2 O, and air-dried.
5. Coverslips are mounted on glass slides using 10 μL Mowiol
4-88 containing 2.5% DABCO (1,4-diazobicyclo-[2.2.2]octane), the number of positive cells is counted, and titers
estimated as described for GFP detection above.
As alphavirus infection causes changes to the cell morphology
(i.e., they round up) microscopic examination can also be applied to
provide an approximate estimate of titers. Similar to fluorescence
and staining (described above), the titers can be estimated by
counting the rounded cells.
3.8.4 RT-PCR-Based
Titer Determination
Another alternative for titer determination is to apply
RT-PCR [36].
1. RNA from SFV stocks is extracted using the RNeasy Kit
(Qiagen).
2. qRT-PCR is applied for sequence amplification within the nsP1
gene of SFV.
3. A serial dilution is made of pSFV plasmid DNA to obtain a
standard curve for SFV-RNA.
4. Mean Cts are applied to a standard curve equation to determine the cDNA copy number in each sample.
5. The total SFV RNA copy number present in the original SFV
sample is calculated by multiplying the cDNA copy number by
the conversion factor specific to each sample considering all
dilutions made and the yield of RNA extraction.
3.9 Evaluation
of Gene Expression
Initial confirmation of recombinant protein expression is recommended before proceeding to immunization studies as it enables
verification of expression levels and the size of gene products.
Expression evaluation can be performed by Western blotting if
antibodies are available against the target protein or engineered
tag fusions. Alternatively, expression can be evaluated by metabolic
labeling of SFV-infected cells with
35 S-methionine.
3.9.1 Western Blotting
1. Host cells (BHK-21, CHO-K1, HEK293) cultured on 6-, 12-,
or 24-well plates are infected with serial dilutions of virus stocks
and incubated for 1–2 days at 37
C.
2. Cells are lysed with 250 μL, 125 μL, and 62.5 μL of lysis buffer
per 6-, 12-, and 24-well plate, respectively, incubated for
10 min on ice and samples are loaded onto 10–12%
SDS-PAGE (polyacrylamide gel electrophoresis) gels.
74
Kenneth Lundstrom
same buffer for 30 min at room temperature and washed three
times with PBS and incubated with a secondary antibody for
30 min at room temperature.
4. Washed coverslips are rewashed three times with PBS, once
more with H 2 O, and air-dried.
5. Coverslips are mounted on glass slides using 10 μL Mowiol
4-88 containing 2.5% DABCO (1,4-diazobicyclo-[2.2.2]octane), the number of positive cells is counted, and titers
estimated as described for GFP detection above.
As alphavirus infection causes changes to the cell morphology
(i.e., they round up) microscopic examination can also be applied to
provide an approximate estimate of titers. Similar to fluorescence
and staining (described above), the titers can be estimated by
counting the rounded cells.
3.8.4 RT-PCR-Based
Titer Determination
Another alternative for titer determination is to apply
RT-PCR [36].
1. RNA from SFV stocks is extracted using the RNeasy Kit
(Qiagen).
2. qRT-PCR is applied for sequence amplification within the nsP1
gene of SFV.
3. A serial dilution is made of pSFV plasmid DNA to obtain a
standard curve for SFV-RNA.
4. Mean Cts are applied to a standard curve equation to determine the cDNA copy number in each sample.
5. The total SFV RNA copy number present in the original SFV
sample is calculated by multiplying the cDNA copy number by
the conversion factor specific to each sample considering all
dilutions made and the yield of RNA extraction.
3.9 Evaluation
of Gene Expression
Initial confirmation of recombinant protein expression is recommended before proceeding to immunization studies as it enables
verification of expression levels and the size of gene products.
Expression evaluation can be performed by Western blotting if
antibodies are available against the target protein or engineered
tag fusions. Alternatively, expression can be evaluated by metabolic
labeling of SFV-infected cells with
35 S-methionine.
3.9.1 Western Blotting
1. Host cells (BHK-21, CHO-K1, HEK293) cultured on 6-, 12-,
or 24-well plates are infected with serial dilutions of virus stocks
and incubated for 1–2 days at 37
C.
2. Cells are lysed with 250 μL, 125 μL, and 62.5 μL of lysis buffer
per 6-, 12-, and 24-well plate, respectively, incubated for
10 min on ice and samples are loaded onto 10–12%
SDS-PAGE (polyacrylamide gel electrophoresis) gels.
74
Kenneth Lundstrom