Basic nucleofector kit for primary mammalian epithelial cells

Silencing of Smad2, Smad3, Smad4, IGFBP7, MAPK1 or MAPK8 gene expression in HRPTECs was achieved by the siRNA technique that has recently been used to study gene function in mammalian cells. The transfection of HRPTECs was carried out by electroporation using the Nucleofection^® system (Amaxa, Koln, Germany), according to the protocols proposed by Amaxa. Briefly, 2 × 10⁶ HRPTECs were resuspended in 100 μl of nucleofector solution (Basic Nucleofector Kit for Primary Mammalian Epithelial Cells, Lonza, Allendale, NJ) containing 100 pmol of double-stranded siRNAs (control, Smad2, Smad3, Smad4, IGFBP7, MAPK1 or MAPK8 siRNAs). After electroporation, 500 μl of pre-warmed cultured medium was added to the cuvette, and the cells were transferred into six-well cultures plates containing prewarmed culture medium. Forty-eight hours after transfection, HRPTECs were serum-starved for an additional 24 h and subsequently stimulated with TGF-β1 (2.5 ng/ml) for the indicated times. The expression of the targeted molecule was monitored by Western blotting or a real-time qRT-PCR. To confirm the efficacy of the reduction in the specific RNA on cells transfected with specific RNA relative the control siRNA, we performed qRT-PCR using HRPTECs transfected with IGFBP7 siRNAs, and showed the data in S1 Fig.

Podocytes were transfected with pmaxGFP encoding green fluorescent protein (Lonza) together with pCR3.1 (Invitrogen) encoding full-length N protein of PUUV or HTNV using nucleofection (Amaxa Nucleofector 2b device, Lonza) with the Basic Nucleofector Kit for primary mammalian epithelial cells (Lonza). Transfection of pmaxGFP together with empty vector (mock) served as control. After eight hours, cells were subjected to live cell imaging or immunofluorescence. All cells expressing maxGFP co-expressed hantaviral N protein. Percentage and viability of cells expressing max GFP together with N protein did not differ compared to cells transfected with pmaxGFP and empty vector. Amount of N protein expression was quantified by determination of the fluorescence levels of 100 transfected cells that were stained for N protein with anti-N protein antibody. Area, mean fluorescence signal, and adjacent background readings were measured for each cell using ImageJ (v1.52e, NIH). The fluorescence levels were calculated with ImageJ as total corrected cell fluorescence (TCCF) of N protein (TCCF = integrated density – (area of selected cell × mean background fluorescence)) [12 (link)].