3.4 Antibody Assay
by PRNT
3.4.1 Preparation
of Growth Medium
(DMEM + 5%
FCS + Antibiotics)
1. Thaw required number of heat-inactivated FCS frozen aliquots
at 4
C and mix well.
2. Filter sterile 25 mL of FBS and 5 mL of 100Â Penicillin/
Streptomycin when adding them to 500 mL of DMEM.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at 2–8
C.
4. Media should be verified that it is not contaminated before
each use by visually inspecting for cloudiness or foreign particles. If contaminated, the media must be discarded.
3.4.2 Preparation
of Maintenance Medium
(DMEM + 3%
FCS + Antibiotics)
1. Thaw required volume of heat-inactivated FCS at 4
C and
mix well.
2. Filter sterile 15 mL of FCS and 5 mL of 100Â Penicillin/
Streptomycin when adding them to 500 mL DMEM.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at 2–8
C.
4. Media should be verified that it is not contaminated before
each use by visually inspecting for cloudiness or foreign particles. If contaminated, the media must be discarded.
3.4.3 Preparation
of Fixative Solution
1. Mix 100 mL of formalin with 900 mL of PBS.
2. Thoroughly mix.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at RT.
3.4.4 Preparation
of Methylene Blue Stock
Solution
1. Dissolve 2.25 g Methylene Blue in 200 mL water and add
0.375 mL 1 M NaOH.
2. Thoroughly mix.
3. Label dark container with appropriate information and record
all information on the appropriate reagent formulation worksheet. Store at RT.
3.4.5 Preparation
of Overlay Solution
1. Dissolve 9.6 g of Avicel Powder in 400 mL of Milli-Q Water.
2. Thoroughly mix and autoclave.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at RT.
3.4.6 Overlaying Media
for Plaque Assay
1. Thaw heat-inactivated FCS, 100Â Penicillin-Streptomycin and
100Â GlutaMAX aliquots at 4
C and mix well.
DNA Vaccine Design and Adjuvant Selection
417
by PRNT
3.4.1 Preparation
of Growth Medium
(DMEM + 5%
FCS + Antibiotics)
1. Thaw required number of heat-inactivated FCS frozen aliquots
at 4
C and mix well.
2. Filter sterile 25 mL of FBS and 5 mL of 100Â Penicillin/
Streptomycin when adding them to 500 mL of DMEM.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at 2–8
C.
4. Media should be verified that it is not contaminated before
each use by visually inspecting for cloudiness or foreign particles. If contaminated, the media must be discarded.
3.4.2 Preparation
of Maintenance Medium
(DMEM + 3%
FCS + Antibiotics)
1. Thaw required volume of heat-inactivated FCS at 4
C and
mix well.
2. Filter sterile 15 mL of FCS and 5 mL of 100Â Penicillin/
Streptomycin when adding them to 500 mL DMEM.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at 2–8
C.
4. Media should be verified that it is not contaminated before
each use by visually inspecting for cloudiness or foreign particles. If contaminated, the media must be discarded.
3.4.3 Preparation
of Fixative Solution
1. Mix 100 mL of formalin with 900 mL of PBS.
2. Thoroughly mix.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at RT.
3.4.4 Preparation
of Methylene Blue Stock
Solution
1. Dissolve 2.25 g Methylene Blue in 200 mL water and add
0.375 mL 1 M NaOH.
2. Thoroughly mix.
3. Label dark container with appropriate information and record
all information on the appropriate reagent formulation worksheet. Store at RT.
3.4.5 Preparation
of Overlay Solution
1. Dissolve 9.6 g of Avicel Powder in 400 mL of Milli-Q Water.
2. Thoroughly mix and autoclave.
3. Label container with appropriate information and record all
information on the appropriate reagent formulation worksheet. Store at RT.
3.4.6 Overlaying Media
for Plaque Assay
1. Thaw heat-inactivated FCS, 100Â Penicillin-Streptomycin and
100Â GlutaMAX aliquots at 4
C and mix well.
DNA Vaccine Design and Adjuvant Selection
417