Quantikine elisa kit

To investigate whether silencing of SRGN mRNA expression can influence myoblast proliferation, human myoblast cultures of 50% confluence were incubated for 24 h with 10 nmol/L SRGN siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) or scramble siRNA (Qiagen) and 8 μL/mL HiPerfect Transfection Reagent (Qiagen) in normal proliferation medium. After another 48 h cell numbers were counted with a Countess™ automated cell counter (Invitrogen) and protein concentrations were measured with a Pierce™ BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). The effect of SRGN silencing on the mRNA expression and secretion of SERPINE1 was investigated in myotubes. The muscle cell cultures were differentiated for 1 day and then incubated with 10 nmol/L SRGN siRNA or scramble siRNA and 16 μL/mL HiPerfect Transfection Reagent in medium containing 2% horse serum for 24 h. After another 48 h the cells were incubated with serum-free medium for 24 h and the concentration of SERPINE1 in the conditioned medium was measured with a Quantikine ELISA Kit (RnD Systems, McKinley Place NE, MN). The silencing efficacy was quantified by reverse transcription polymerase chain reaction after 24, 48 and 96 h and untreated controls and controls incubated with only HiPerfect reagent were included.

To examine the concentration of growth factors secreted by hBMVEC, cells were grown to confluency and treated with 10 μM mephedrone in growth factor-free medium for 24 h. Cell culture supernatant was briefly centrifugated (5 min at 2000g) to pellet cell debris and then analyzed using Human PDGF BB Elisa kit (Invitrogen Cat No BMS2071) or vascular endothelial growth factor A (VEGF-A) Quantikine Elisa kit (RnD Systems Cat No DVE00) as described in the manufacturer’s protocol.