Anti-Neu5Gc Antibody Kit Protocol - Flow Cytometry
Prepare 0.5% Neu5Gc Assay Blocking Solution in PBS (diluent buffer).
The following tubes should be set up for flow cytometry analysis: 
* Three (3) tubes containing cells to be examined.
If choosing to use different dilutions of primary antibody, as noted in step 3, more tubes may be needed.
* Three (3) tubes containing positive control cells.
* Three (3) tubes containing negative control cells. 
Each set of tubes to be analyzed should receive an antibody treatment as follows:
* Tube 1 will contain cells that will receive no antibody, to be used as unstained control (optional but highly recommended).
* Tube 2 will contain cells that receive Control Antibody.
* Tube 3 will contain cells that receive Primary Antibody.
- Note: if this is the first time running this experiment, optimize by using different dilutions of primary antibody.
More tubes containing sample will be needed.
Wash cells by adding 1 ml cold PBS, then gently centrifuge at 4°C.
Carefully remove supernatant and discard.
Gently resuspend cells in 100 μl of the appropriate diluted antibody as outlined above.
Incubate cells on ice for at least 1 hour. 
Wash cells as above. 
Gently resuspend cells in 100 μl Secondary Antibody in diluent buffer, and incubate for 1 hour on ice.
Wash cells as before.
Resuspend cell pellet in 400 μl of diluent buffer. 
Run cells through the flow cytometer.
