Hi, The problem is with non-specific binding of tetramer to dead cells. We usually stain with annexin-V to exclude non-specific binding of tetramer. I find it helpful to set up a plot of annexin-V versus tetramer gated on CD8+ population to calculate the fraction of "true" tetramer+/CD8+ cells. This may not be necessary with fresh PBMC's but with cultured T cells it can increase the sensitivity considerably. Good luck, Nick Haining W. Nicholas Haining, B.M., B.Ch. Pediatric Oncology/Hematology Dana Farber Cancer Institute -----Original Message----- From: Michal Abel [SMTP:abelm@necker.fr] Sent: Monday, February 11, 2002 1:10 PM To: Cytometry Mailing List Subject: Negative gating and rare events Hello, I'm working with HLA class I tetramers. I've heard that negative gating can help reducing background staining in rare event detection. Can someone more experienced tell me if this is true and if so, why ? Thanks in advance. -- Michal Abel, MD, PhD Laboratory of Clinical Immunology INSERM U25 Necker Hospital, 161 Rue de Sèvres. Paris tel: 33 1 44 49 52 23 fax: 33 1 44 49 53 74
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