suspension back into tube through strainer using a serological
pipette. Place cells on ice.
7. Determine concentration of samples following dilution (e.g.,
tenfold) and addition of appropriate live/dead stain (e.g., trypan blue) by counting cells using a hemocytometer or automated cell counter (see Note 8).
3.4 Preparation
of Sample Dilutions
and Stimulants
1. Using measured cell concentrations, calculate appropriate
amount of cells from each sample to add to 1 mL of R10
media to obtain a 4 Â 10
6 cells/mL cell suspension (see Note
9). Vortex cell suspension gently and add appropriate volume
of each sample to a single well of a multichannel reservoir. Once
all cell samples have been added, dispense 1 mL of R10 media
to each well to adjust cells to a final concentration of
4 Â 10
6 cells/mL.
2. Thaw stimulants at room temperature. Prepare following
2Â stimulant solutions by pipetting the appropriate amount
of stimulant to a conical tube containing R10 media. 0.2 mL of
each solution will be required for each sample.
(a) Media alone: 2 μL DMSO per mL of R10.
(b) 2Â CD8 epitope solution (4 μg/mL SIINFEKL): 2 μL
2 mg/mL SIINFEKL per mL of R10.
(c) 2Â CD4 epitope solution (4 μg/mL ISQAVHAAHAEINEAGR): 2 μL 2 mg/mL ISQAVHAAHAEINEAGR per
mL of R10.
(d) Positive control (2Â conA solution (10 μg/mL)): 2 μL of
5 mg/mL conA per mL of R10. ConA is a mitogen that
will induce IFN-γ secretion in a non-antigen-specific manner (see Note 10).
3. Dump R10 media from blocked ELISpot plates within biosafety cabinet. Immediately add 100 μL of stimulants per well
of plates according to plate layout (see Fig. 2 for an example of a
plate layout).
4. Resuspend cell suspension in multichannel reservoir by pipetting up and down 10–15 times. Add 100 μL of cells per well of
plates according to plate layout (see Note 11).
5. Shake cells gently on orbital shaker for ~60 s to evenly disperse
cells within well and place plates in 37
C humidified incubator
with 5% CO 2 overnight for ~20 h (see Note 12).
3.5 Plate
Development
1. Dilute the biotinylated detection antibody with diluent buffer
to a final concentration of 1 μg/mL. Each plate will require
10 mL.
2. Remove the plates from the incubator and dump cell suspension to a plastic container. With a multichannel pipette, add
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Bassel Akache and Michael J. McCluskie
pipette. Place cells on ice.
7. Determine concentration of samples following dilution (e.g.,
tenfold) and addition of appropriate live/dead stain (e.g., trypan blue) by counting cells using a hemocytometer or automated cell counter (see Note 8).
3.4 Preparation
of Sample Dilutions
and Stimulants
1. Using measured cell concentrations, calculate appropriate
amount of cells from each sample to add to 1 mL of R10
media to obtain a 4 Â 10
6 cells/mL cell suspension (see Note
9). Vortex cell suspension gently and add appropriate volume
of each sample to a single well of a multichannel reservoir. Once
all cell samples have been added, dispense 1 mL of R10 media
to each well to adjust cells to a final concentration of
4 Â 10
6 cells/mL.
2. Thaw stimulants at room temperature. Prepare following
2Â stimulant solutions by pipetting the appropriate amount
of stimulant to a conical tube containing R10 media. 0.2 mL of
each solution will be required for each sample.
(a) Media alone: 2 μL DMSO per mL of R10.
(b) 2Â CD8 epitope solution (4 μg/mL SIINFEKL): 2 μL
2 mg/mL SIINFEKL per mL of R10.
(c) 2Â CD4 epitope solution (4 μg/mL ISQAVHAAHAEINEAGR): 2 μL 2 mg/mL ISQAVHAAHAEINEAGR per
mL of R10.
(d) Positive control (2Â conA solution (10 μg/mL)): 2 μL of
5 mg/mL conA per mL of R10. ConA is a mitogen that
will induce IFN-γ secretion in a non-antigen-specific manner (see Note 10).
3. Dump R10 media from blocked ELISpot plates within biosafety cabinet. Immediately add 100 μL of stimulants per well
of plates according to plate layout (see Fig. 2 for an example of a
plate layout).
4. Resuspend cell suspension in multichannel reservoir by pipetting up and down 10–15 times. Add 100 μL of cells per well of
plates according to plate layout (see Note 11).
5. Shake cells gently on orbital shaker for ~60 s to evenly disperse
cells within well and place plates in 37
C humidified incubator
with 5% CO 2 overnight for ~20 h (see Note 12).
3.5 Plate
Development
1. Dilute the biotinylated detection antibody with diluent buffer
to a final concentration of 1 μg/mL. Each plate will require
10 mL.
2. Remove the plates from the incubator and dump cell suspension to a plastic container. With a multichannel pipette, add
530
Bassel Akache and Michael J. McCluskie