Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

ABSTRACT

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.