200 μL of water to each well and incubate the plate at room
temperature for 5 min (see Note 13).
3. Dump the water and again with a multichannel pipette, wash
the plates 4Â with 200 μL of DPBS per well.
4. Tap the plates on a paper towel to get rid of the excess DPBS
and add 100 μL of the detection antibody solution to each well.
Incubate the plates for 2 h at room temperature.
5. Dilute the 1000Â streptavidin-HRP 1:1000 with diluent
buffer to the appropriate final concentration. Each plate will
require 10 mL.
6. Dump the solution from the plates and with a multichannel
pipette, wash the plates 5Â with 200 μL of DPBS per well.
7. Tap the plates on a paper towel to get rid of the excess DPBS
and add 100 μL of the streptavidin-HRP solution to each well.
Incubate the plates for 1 h at room temperature.
8. Just prior to the completion of the above incubation step,
prepare the AEC substrate solution (see Note 14). The AEC
substrate set contains separate AEC substrate and chromagen
solutions. For each plate, add 10 mL of AEC substrate solution
and 200 μL of the chromagen solution to a conical tube. Invert
tube a few times to mix solution and keep at room temperature
in the dark until ready to use.
Fig. 2 Example of plate layout
Quantification of Antigen-Specific T cells
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