IFNγ ELISpot

Coating Plates:

1. ELISpot Kit used: BD551083
2. Dilute capture antibody in 1X PBS (Use 50µl/10ml, 1:200 for C_F of 5µg/ml)
3. Add 100µl of diluted antibody solution to each well of the ELISpot plate. 
4. Store plates at 4°C overnight

Blocking:

1. Discard capture antibody by flicking plate and then tapping on paper towel. 
2. Wash wells 1x with complete RPMI media.
3. Add 200µl/well of complete RPMI and incubate for 2h at room temperature.

Cell Activation:

1. Discard blocking media by flicking plate and then tapping on paper towel.
2. Add freshly harvested/processed cells to wells in 100µl media. 
3. Add stimulating peptide (1µg/well) to wells in 100µl media. 
4. Incubate overnight (ca. 20h)

Detection Antibody:

1. Discard well contents by flicking plate and then tapping on paper towel.
2. Wash wells 2x with deionized water. Allow wells to soak for 3-5min each time. 
3. Wash wells 3x with PBS + 0.05% Tween-20. Discard washing buffer by flicking plate and then tapping.
4. Dilute Detection Antibody in PBS + 10% FBS. (Use 40µl/10ml, 1:250 for C_F of 2µg/ml)
5. Add 100µl of diluted antibody solution to each well of the ELISpot plate. 
6. Incubate for 2h at room temperature.

Enzyme Conjugate:

1. Discard detection antibody solution by flicking plate and then tapping on paper towel.
2. Wash wells 3x with PBS + 0.05% Tween-20. Discard washing buffer by flicking plate and then tapping. Allow plates to soak for 1-2min each time. 
3. Dilute Streptavidin-HRP in PBS + 10% FBS. (Use 100µl/10ml, 1:100)
4. Add 100µl of enzyme conjugate solution to each well of the ELISpot plate. 
5. Incubate for 1h at room temperature.

Substrate:

1. Discard enzyme conjugate solution by flicking plate and then tapping.
2. Wash wells 4x with PBS + 0.05% Tween-20. Discard washing buffer by flicking plate and then tapping. Allow plates to soak for 1-2min each time. 
3. Wash wells 2x with PBS. Discard washing buffer by flicking plate and then tapping. 
4. Add 100µl of Final Substrate Solution (AEC Substrate Set; BD 551951) to each well.
5. Monitor spot development from 1-8min
6. Stop substrate reaction by washing wells with DI water.
7. Air-dry plate at room temperature in the hood.