English to Russian Science Biology (-tech,-chem,micro-) иммунология

Fibroblast gel contraction assay

NHLF’s are expanded up to 90% confluence in FGM-2 growth media (Lonza) supplemented with 2% FBS (plus SingleQuotTM growth factors). Fibroblasts are harvested (Trypsin/EDTA) and suspended at 1x106 per ml in serum free media. Based on the cell contraction assay kit (Cell Biolabs) a cell lattice is prepared by mixing 1 part cell suspension + 4 parts collagen gel solution as per kit instructions. 0.9ml aliquots of the collagen lattice is added to 1.5 ml centrifuge tubes and treated with final assay concentrations of test compound. 250µl of compound treated lattice is then pipetted into each well of a 48 well tissue culture plate (triplicate per test concentration). The plate is incubated for 90 min (37 C/ 5%CO2/95%O2) to allow the gels to polymerize. 250µl of serum free media containing final assay concentrations of test compound are then added to each corresponding gel. After further 30 min incubation, the gels are stimulated with TGFβ1 (10ng/ml; R&D Systems). Following a 24 hr (37 C/ 5%CO2/95%O2) incubation period, each individual gel is removed and weighed on a precision balance. The effect of the test compound at each concentration is expressed as the percent reversal of the TGFβ1 induced contraction relative to the vehicle treated basal contraction.

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