I'm trying to set up a template on a FACSCalibur for the identification of T helper and cytotoxic T cells. I'm using whole blood lysis with BD FACSLyse and BDs CD3-FITC and CD4-PE. I have two problems:
1)On the FSC Vs SSC dot plot I can only see the lymphocyte population.
2)On the fluorescence plots I can't see any positively staining populations. I'm using the recommended Ab staining of 20ul per 10million cells.
This may seem like a simple problem but before I continue I wanted a second opinion. Is is just a case of titrating the Ab's i.e adding more, or would the whole blood lysis be at fault? Is there anything I could do to improve this to get other populations on the FSC Vs SSC plot and to give a comfortable separation between the negative and positive staining populations on the flourescence plots. I'm not really a flow expert!!
Thanks for your help!