9. Dump the solution from the plates. With a multichannel
pipette, wash the plates 5Â with 200 μL of DPBS per well.
10. Tap the plates on a paper towel to get rid of the excess DPBS
and add 100 μL of the AEC solution to each well. Incubate
plates for 20 min at room temperature (see Note 15).
11. Stop the reaction by submerging the plate in a large plastic
container being continuously filled with running tap water in a
sink. Quickly remove and flick the plate to dump out the water
in the sink. Resubmerge the plate ensuring that all wells are
filled with the water. Repeat 4–6 times.
12. Remove the underdrain (plastic covering on bottom of plate)
from the plate, and wash the plate again by submerging a few
times as above in the water container (see Note 16).
13. Allow plates to dry at room temperature overnight.
Fig. 3 IFN-γ + spot forming cells from splenocytes of immunized mice. C57Bl/6
mice were immunized intramuscularly with OVA (10 μg) alone or adjuvanted with
alum (40 μg) + CpG 1826 (10 μg) on days 0 and 21. Spleens were collected on
Day 28 with splenocytes analyzed for OVA-specific CD4 T cells by IFN-γ ELISpot.
Images of representative wells are shown in Panels (a)–(d). Cells from a mouse
immunized with OVA alone stimulated with media or 2 μg/mL ISQAVHAAHAEINEAGR in Panels (a) and (b), respectively. Cells from a mouse immunized with
OVA + alum/CpG stimulated with media or 2 μg/mL ISQAVHAAHAEINEAGR in
Panels (c) and (d), respectively
532
Bassel Akache and Michael J. McCluskie
pipette, wash the plates 5Â with 200 μL of DPBS per well.
10. Tap the plates on a paper towel to get rid of the excess DPBS
and add 100 μL of the AEC solution to each well. Incubate
plates for 20 min at room temperature (see Note 15).
11. Stop the reaction by submerging the plate in a large plastic
container being continuously filled with running tap water in a
sink. Quickly remove and flick the plate to dump out the water
in the sink. Resubmerge the plate ensuring that all wells are
filled with the water. Repeat 4–6 times.
12. Remove the underdrain (plastic covering on bottom of plate)
from the plate, and wash the plate again by submerging a few
times as above in the water container (see Note 16).
13. Allow plates to dry at room temperature overnight.
Fig. 3 IFN-γ + spot forming cells from splenocytes of immunized mice. C57Bl/6
mice were immunized intramuscularly with OVA (10 μg) alone or adjuvanted with
alum (40 μg) + CpG 1826 (10 μg) on days 0 and 21. Spleens were collected on
Day 28 with splenocytes analyzed for OVA-specific CD4 T cells by IFN-γ ELISpot.
Images of representative wells are shown in Panels (a)–(d). Cells from a mouse
immunized with OVA alone stimulated with media or 2 μg/mL ISQAVHAAHAEINEAGR in Panels (a) and (b), respectively. Cells from a mouse immunized with
OVA + alum/CpG stimulated with media or 2 μg/mL ISQAVHAAHAEINEAGR in
Panels (c) and (d), respectively
532
Bassel Akache and Michael J. McCluskie