I am posting a couple of questions from an investigator who wants to look at annexin V staining on the surface of antigen-specific T cells [KJ1-26+] in the blood of mice. These cells were adoptively transferred two days prior to a single injection of OVA. Her protocol includes (1) staining whole mouse blood for CD4 and KJ1-26 TCR, (2) lysing RBCs with an ammonium chloride buffer solution, and (3) staining for annexin V and sytox green, using the Vybrant apoptosis kit from Molecular Probes. Her initial findings indicated a high degree of annexin V staining on KJ1-26+CD4+ cells in the blood (>50%)--even in mice that were adoptively transferred with KJ1-26+ cells but not injected with OVA. In contrast, she found only 20% of the KJ1-26+CD4+ cells in the spleen that stained positive for annexin V. Splenic RBCs were lysed, using the same ammonium chloride buffer, before she stained for CD4 and KJ1-26. Then, she used the Vybrant apoptosis kit to stain for annexin V. The questions are: what is causing the high background in the blood? Is the high level of annexin V due to some technical error? And how can she reduce this background level? Any comments? Any suggestions? Thanks in advance, Julie Oughton Oregon State University EHSC flow cytometry lab
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