T0020001

Pure compounds were tested against human fibroblast cells (HaCaT) using a standard MTT cell viability assay [44 (link)]. HaCaT human fibroblast cells (AddexBio T0020001, San Diego, CA, USA) were maintained in growth media: DMEM (Invitrogen 11965-092, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen 16000-044), 1% penicillin/streptomycin (Invitrogen 15140-122), 1% sodium pyruvate (Invitrogen 11360), and 1% Glutamax-1 (Invitrogen 35050-061). 96-well plates were coated with 0.01% collagen overnight and rinsed with PBS prior to seeding with 25 × 104 cells/mL HaCaT cells and allowed to adhere overnight. After 24 h, compounds were added in triplicate at nine, 3x dilutions from 100 μM final concentration in growth media. Plates were incubated for 72 h at 37 °C in a 5% CO₂/95% air humidified atmosphere after which time the media was removed and MTT was added in a RPMI phenol red free media (Invitrogen 11835-030). The MTT was removed after 3 h, formazan crystals were solubilized with 200 μL of isopropanol and plates were read on an i3 spectrophotometer (Molecular Devices, San Jose, CA, USA) at 570 nm for formazan and 690 nm for background subtraction. EC₅₀ values were calculated by fitting the data in the GraphPad Prism software (version 5.04).