Human tgf β1 quantikine elisa kit

Human SCF Quantikine ELISA kit (R&D Systems, #DCK00) and the Human TGF-β1 Quantikine ELISA kit (R&D Systems, #DB100B) were used for cytokine quantification following the manufacturer’s recommendations.

Adult human skin fibroblast cells were purchased from Lonza (Lonza, USA, cat. # CC-2511) and expended in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM; Gibco -Invitrogen, USA) supplemented with 20% fetal bovine serum (FBS; Gibco -Invitrogen, USA), 100 μM 2-mercaptoethanol (Sigma-Aldrich, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco -Invitrogen, USA) and incubated at standard conditions (humidified atmosphere, 5% CO2, 37°C) for enough expansion. The blood (10 ml) was collected in EDTA-tube from 5 healthy volunteers and centrifuged at 400 g for 15 min and the supernatant that contained platelet rich plasma (PRR) was collected in a new tube. Then, the PRR were incubated with 25 mM CaCl₂ at room temperature for 1 hour in order to activate the platelets. The activated PRR was then centrifuged at 2500g for 15 min to collect soluble releasate of PRCR that was used in the following experiments. The concentrations of TGF-β1 in whole blood and PRCR preparations were measured using an ELISA kit (Human TGF-β1 Quantikine ELISA Kit, R&D system, Cat. # DB100B). In total, 7 samples of blood samples or PRCR were used to obtain TGF-β1 measurements.