HLMFs were grown on T25 flasks and serum starved for 24 h prior to the experiment. The HLMFs were either left unstimulated or stimulated for 24 h with TGFβ1 (10 ng/ml), in the presence of 0.1% DMSO control, TRAM-34 (200 nM) or ICA-17043 (100 nM). The cells were detached using 0.1% trypsin/0.1% EDTA, washed then fixed and permeabilized in 4% paraformaldehyde plus 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA), respectively, for 20 min on ice. Myofibroblasts were labelled with FITC-conjugated mouse monoclonal anti-αSMA (Sigma-Aldrich, St. Louis, MO, USA), indirectly labelled with FITC and isotype control FITC-conjugated mouse IgG2a. Secondary antibodies labelled with FITC (F0313, Dako, Glostrup, Denmark) were applied. Analysis was performed using single colour flow cytometry on a FACScan (BD, Oxford, UK).
Fitc conjugated mouse monoclonal anti αsma
Manufactured by Merck Group
Sourced in United States
FITC-conjugated mouse monoclonal anti-αSMA is a laboratory reagent used for the detection and quantification of alpha-smooth muscle actin (αSMA) in biological samples. It is a fluorescently labeled antibody that specifically binds to αSMA, a cytoskeletal protein expressed in smooth muscle cells and myofibroblasts.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using fitc conjugated mouse monoclonal anti αsma
1
Myofibroblast Characterization by Flow Cytometry
2
TGFβ1-Induced Myofibroblast Phenotype
3
Analyzing Myofibroblast LXA4 Receptor Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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 TGF-β1 (10 ng/ml) (R&D Systems, Oxford, U.K.).
To determine the expression of the LXA4 receptor ALXR, HLMFs were detached using 0.1% trypsin/0.1% EDTA and gated using fibroblast surface Ag Thy-1 (Merck, Hertfordshire, U.K.). Myofibroblasts were then labeled with allophycocyanin-conjugated mouse monoclonal anti–formyl-peptide receptor-like 1 (FPRL1; also known as ALXR) (R&D Systems) or allophycocyanin -conjugated isotype control IgG2b Ab (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 and 10−8 mol. 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, U.K.) for 20 min on ice. Myofibroblasts were labeled with either: FITC-conjugated mouse monoclonal anti-αSMA (Sigma-Aldrich) or isotype control FITC-conjugated mouse IgG2a; secondary Abs labeled with FITC were applied if appropriate. Analysis was performed using single-color flow cytometry on an FACScan (BD Biosciences, Oxford, U.K.).
To determine the expression of the LXA4 receptor ALXR, HLMFs were detached using 0.1% trypsin/0.1% EDTA and gated using fibroblast surface Ag Thy-1 (Merck, Hertfordshire, U.K.). Myofibroblasts were then labeled with allophycocyanin-conjugated mouse monoclonal anti–formyl-peptide receptor-like 1 (FPRL1; also known as ALXR) (R&D Systems) or allophycocyanin -conjugated isotype control IgG2b Ab (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 and 10−8 mol. 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, U.K.) for 20 min on ice. Myofibroblasts were labeled with either: FITC-conjugated mouse monoclonal anti-αSMA (Sigma-Aldrich) or isotype control FITC-conjugated mouse IgG2a; secondary Abs labeled with FITC were applied if appropriate. Analysis was performed using single-color flow cytometry on an FACScan (BD Biosciences, Oxford, U.K.).
4
Immunofluorescent Staining of Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetone fixed fibroblasts on chamber slides were incubated with 10% normal mouse serum for 45 minutes followed by FITC conjugated mouse monoclonal anti-α-SMA (Sigma) for 1 hour at room temperature. After washing, the slides were mounted with 50% glycerol in PBS, and then observed using a Zeiss AxioVert 200 fluorescence microscope. DAPI was used for nuclear staining. The images were recorded by an attached digital camera. 200 cells were counted in each slide, and the percent of α-SMA positively stained cells was calculated.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!