calcium phosphate kit profection mammalian transfection system cat # e1200  (Promega)

Journal: FASEB BioAdvances

Article Title: Low pH up‐regulates interleukin‐6 mRNA in L6‐G8C5 rat skeletal muscle cells independent of pH sensing by SNAT2(SLC38A2) transporters

doi: 10.1096/fba.2021-00088

Figure Lengend Snippet: Effect of siRNA silencing of expression of the SNAT2(SLC38A2) amino acid transporter on interleukin‐6 mRNA in L6‐G8C5 myoblasts. (A) L6 myoblasts were cultured in DMEM growth medium for 24 h and then transfected with 30 nM scrambled control siRNA (Scr) or silencing anti‐SNAT2 siRNA (Sil) for 16 h. Negative control cultures were also treated with transfection agent only, or no additions. The medium was then replaced with growth medium for 24 h and incubated for a further 6 h in MEM medium at pH 7.4 with 2 mM l ‐glutamine with 2% dialysed foetal bovine serum. Pooled data are shown from n = 4 independent experiments. (B, C) L6 myoblasts were transfected or treated with negative transfection controls as in (A) but the subsequent 6‐h incubations in MEM at pH 7.4 were performed either (B) in absence of ionomycin, or (C) with 0.5 µM ionomycin. Pooled data are shown from n = 4 independent experiments

Article Snippet: After overnight incubation, the GM was discarded and 1 ml of fresh GM was added to the wells and the cells were transfected with anti‐SNAT2 SIL siRNA or SCR control using a Profection Calcium Phosphate Transfection Kit (Promega).

Techniques: Expressing, Cell Culture, Transfection, Control, Negative Control, Incubation