Complement-dependent cytotoxicity for negative selection at the mRNA level.
Complement-Dependent Cytotoxicity (CDC) is a common technique used for isolating and characterizing cell populations. However, the molecular events resulting from CDC-mediated cell injury remain obscure. In order to use CDC as a selection procedure for studies at the RNA level, we examined if CDC is associated with rapid degradation of RNAs from target cells without affecting the stability and viability of RNAs of non-target cells. Using a model of anti-CD3-mediated CDC, we show that T cell-specific RNAs were absent immediately after CDC. However, ribosomal RNAs and mRNAs from non-targeted cells (non-T cells) were not affected by CDC. Our results indicate that CDC is associated with rapid degradation of only target cell RNAs, validating CDC as a method for cell isolation without interfering with further studies at the RNA level.
Gill
N S
NS
Department of Medicine University of Alberta, Edmonton, Canada.
Madrenas
J
J
Halloran
P F
PF
D000911:Antibodies, Monoclonal; D015152:Blotting, Northern; D017252:CD3 Complex / Q000276:immunology; D002452:Cell Count; D003165:Complement System Proteins / Q000276:immunology*; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D012333:RNA, Messenger / Q000276:immunology*; D012337:RNA, Ribosomal, 18S / Q000276:immunology; D012339:RNA, Ribosomal, 28S / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S; D016693:Receptors, Antigen, T-Cell, alpha-beta; D003165:Complement System Proteins