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 .
