Directly Coupled Antibody Titration Protocol

1. In microtiter plate, serially dilute antibody in buffer.

Serial Dilutions:
In first well add 32uL of buffer. In next 6 wells add 20uL of buffer.
To first well add 8uL of antibody. Mix well and discard tip.
With clean tip, transfer 20uL from first well into second well. Mix well and discard tip.
Continue 2-fold serial dilution in this fashion until there is 40uL of diluted antibody in well #7.
Serial dilution is now complete as follows: 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320.

2. Prepare cells for staining by resuspending them at 5x10^6/mL, and put 100uL of cells into test tubes (5x10^5 cells total).

3. Add 10uL of appropriate antibody dilution to tubes according to the following schedule:

Tube 1 = cells alone
Tube 2 = 10uL antibody neet (no dilution)
Tube 3 = 10uL 1:5 dilution
Tube 4 = 10uL 1:10 dilution
Tube 5 = 10uL 1:20 dilution
Tube 6 = 10uL 1:40 dilution
Tube 7 = 10uL 1:80 dilution
Tube 8 = 10uL 1:160 dilution
Tube 9 = 10uL 1:320 dilution

4. Incubate for 20minutes at 4C in the dark.

5. Wash 2x in buffer.

6. Analyze fresh or fix in 1% paraformaldehyde