Hi,

From what I'm seeing in the literature most people go with an initial enrichment with a MACS separator using an antibody against the labelled fluorophore of the Cd1d tetramer. So in my case the Cd1d tetramer we use is in APC- that would mean that I would have to get anti APC microbeads right? I noticed most scientist stain these cells with either TCRb or CD3e only. I have both in my flow panel TCRb-FITC and CD3e V500. Is there a preference for one over the other?

Just to be sure these are these the correct steps?

- Obtain whole lung cells

- Stain with CD16/CD32 mAb for non-specific binding for 10 min at 4C

-Add APC-labelled Cd1d tetramers

- Incubate anti-APC MicroBeads and get iNKT based off positive selection

- Then just do simple staining: viability dye, TCRb and CD3e or both?

I would appreciate anyone who is working with this type of cells in sorting experiments to please provide some guidance.

Thanks