HLMFs were grown in T25 flasks and serum-starved for 24 h prior to experiments. The myofibroblasts were incubated for 24 h and either left unstimulated or stimulated with TGFβ1 (10 ng/ml)(R&D Systems, Oxford, UK).
To determine the expression of the LXA4 receptor ALXR, HLMFs were detached using 0.1% trypsin/0.1% EDTA and gated using fibroblast surface antigen, Thy-1 (Merck, Hertforshire, UK). Myofibroblasts were then labelled with APC-conjugated mouse monoclonal anti-FPRL1 (Formyl-peptide receptor-like 1 [FRPL1], also known as ALXR)(R&D Systems) or APC-conjugated isotype control IgG2b antibody (R&D Systems).
To study the inhibitory effects of LXA4 on αSMA expression, HLMFs were incubated in the presence of serum-free medium alone or 0.1% ethanol vehicle control or LXA4 at 10−10 M and 10−8 M. Cells were detached using 0.1% trypsin/0.1% EDTA, washed, then fixed and permeabilized in 4% paraformaldehyde plus 0.1% saponin (Sigma-Aldrich, Poole, Dorset, UK) for 20 minutes on ice. Myofibroblasts were labelled with either: FITC-conjugated mouse monoclonal anti-αSMA (Sigma) or isotype control FITC-conjugated mouse IgG2a; secondary antibodies labelled with FITC were applied if appropriate. Analysis was performed using single colour flow cytometry on a FACScan (BD biosciences, Oxford, UK).
To determine the expression of the LXA4 receptor ALXR, HLMFs were detached using 0.1% trypsin/0.1% EDTA and gated using fibroblast surface antigen, Thy-1 (Merck, Hertforshire, UK). Myofibroblasts were then labelled with APC-conjugated mouse monoclonal anti-FPRL1 (Formyl-peptide receptor-like 1 [FRPL1], also known as ALXR)(R&D Systems) or APC-conjugated isotype control IgG2b antibody (R&D Systems).
To study the inhibitory effects of LXA4 on αSMA expression, HLMFs were incubated in the presence of serum-free medium alone or 0.1% ethanol vehicle control or LXA4 at 10−10 M and 10−8 M. Cells were detached using 0.1% trypsin/0.1% EDTA, washed, then fixed and permeabilized in 4% paraformaldehyde plus 0.1% saponin (Sigma-Aldrich, Poole, Dorset, UK) for 20 minutes on ice. Myofibroblasts were labelled with either: FITC-conjugated mouse monoclonal anti-αSMA (Sigma) or isotype control FITC-conjugated mouse IgG2a; secondary antibodies labelled with FITC were applied if appropriate. Analysis was performed using single colour flow cytometry on a FACScan (BD biosciences, Oxford, UK).