3.1 Antibody Coating
1. Determine the number of plates required to test desired samples (each sample is usually tested in duplicate).
2. Prepare sufficient quantities of coating solution (~10 mL per
plate) by diluting capture antibody to a final concentration of
15 μg/mL in DPBS (see Note 3).
3. Add 15 μL of 35% ethanol per well of the PVDF ELISpot plate
and incubate for 1 min.
4. Wash each plate well with 200 μL sterile water 5Â using multichannel pipette. Dump wash solutions by flicking plates over a
wide plastic container within the biosafety cabinet to maintain
the plate’s sterility. Tap plate on paper towel after final wash to
remove excess water and add 100 μL of coating solution per
well (see Note 4).
5. Incubate overnight for ~16–20 h at 2–8
C.
3.2 Blocking
of Plates
1. On the following day, wash the plates 5Â with 200 μL per well
of DPBS solution using multichannel pipette (see Note 5).
Again, washes are dumped within the biosafety cabinet and
plates are tapped on paper towel after final wash to remove
excess DPBS.
2. Add 200 μL of R10 to each well and incubate at room temperature for at least 30 min prior to addition of cells and stimulants
prepared below.
3.3 Spleen Collection
and Processing
1. Anesthetize mouse (previously immunized with adjuvanted
ovalbumin vaccine formulation) and euthanize by cervical dislocation (see Note 6).
2. Place mouse on counter, right flank down. Spray left flank with
70% ethanol.
3. Make small incision with surgical scissors across left flank (~1/3
of the way from base of tail to neck) to access abdominal cavity.
4. Locate spleen and gently pull out in an exterior direction
through the incision using tweezers. Detach entire spleen
using surgical scissors and place in 50 mL conical tube containing 5 mL of sterile chilled R10 media and place spleen on ice
(see Note 7).
5. Pour contents of tube (i.e., spleen and R10 media) in a sterile
petri dish. Homogenize tissue by placing spleen in between the
frosted edges of two sterilized microscope slides and sliding
them back and forth gently. Continue rubbing the slides
against each other until the entire tissue has been reduced to
cellular suspension.
6. Place 70 μm cell strainer on same 50 mL conical tube that
contained spleen in step 4 above and transfer splenocyte
Quantification of Antigen-Specific T cells
529
1. Determine the number of plates required to test desired samples (each sample is usually tested in duplicate).
2. Prepare sufficient quantities of coating solution (~10 mL per
plate) by diluting capture antibody to a final concentration of
15 μg/mL in DPBS (see Note 3).
3. Add 15 μL of 35% ethanol per well of the PVDF ELISpot plate
and incubate for 1 min.
4. Wash each plate well with 200 μL sterile water 5Â using multichannel pipette. Dump wash solutions by flicking plates over a
wide plastic container within the biosafety cabinet to maintain
the plate’s sterility. Tap plate on paper towel after final wash to
remove excess water and add 100 μL of coating solution per
well (see Note 4).
5. Incubate overnight for ~16–20 h at 2–8
C.
3.2 Blocking
of Plates
1. On the following day, wash the plates 5Â with 200 μL per well
of DPBS solution using multichannel pipette (see Note 5).
Again, washes are dumped within the biosafety cabinet and
plates are tapped on paper towel after final wash to remove
excess DPBS.
2. Add 200 μL of R10 to each well and incubate at room temperature for at least 30 min prior to addition of cells and stimulants
prepared below.
3.3 Spleen Collection
and Processing
1. Anesthetize mouse (previously immunized with adjuvanted
ovalbumin vaccine formulation) and euthanize by cervical dislocation (see Note 6).
2. Place mouse on counter, right flank down. Spray left flank with
70% ethanol.
3. Make small incision with surgical scissors across left flank (~1/3
of the way from base of tail to neck) to access abdominal cavity.
4. Locate spleen and gently pull out in an exterior direction
through the incision using tweezers. Detach entire spleen
using surgical scissors and place in 50 mL conical tube containing 5 mL of sterile chilled R10 media and place spleen on ice
(see Note 7).
5. Pour contents of tube (i.e., spleen and R10 media) in a sterile
petri dish. Homogenize tissue by placing spleen in between the
frosted edges of two sterilized microscope slides and sliding
them back and forth gently. Continue rubbing the slides
against each other until the entire tissue has been reduced to
cellular suspension.
6. Place 70 μm cell strainer on same 50 mL conical tube that
contained spleen in step 4 above and transfer splenocyte
Quantification of Antigen-Specific T cells
529