Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.
The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.
M
M
Majumdar
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India.
R
R
Ratho
Y
Y
Chawla
M P
MP
Singh
D002470:Cell Survival / Q000502:physiology; D002498:Centrifugation / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering