α-SMA (ACTA2) Assay Kit

Alpha-SMA assay as a key marker of differentiated myofibroblasts and tissue fibrosis

This cell-based assay is designed to measure the expression level of α-SMA (α-Smooth Muscle Actin, also known as ACTA2), a key marker of differentiated myofibroblasts and tissue fibrosis.

After tissue injury, TGF-β locally released by inflammatory cells activates resident fibroblasts or quiescent HSCs (hepatic stellate cells). This leads to their differentiation into contractile and secretory myofibroblasts, whose role is to migrate into the damaged tissue and synthetize ECM (extracellular matrix) components to repair the wound.

Myofibroblasts are characterized by de novo expression of α-SMA, which is incorporated into actin stress fibers and confers a high contractile activity to the cells.

Chronic tissue injury and inflammation lead to persistent de novo formation of myofibroblasts (α-SMA+) and excessive contraction and deposition of ECM, eventually leading to tissue fibrosis (hepatic, pulmonary, renal, cardiac or dermal). Inhibiting myofibroblast differentiation is therefore a promising therapeutic strategy to treat fibrotic disorders.

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s Alpha-SMA assay is based on a TR-FRET sandwich immunoassay format comprising two antibodies, one labeled with Eu3+cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to Alpha-SMA, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 96- or 384-well low volume microplate format (20 µL final), but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assay can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous Alpha-SMA can be quantitatively detected using the HTRF Alpha-SMA cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol:

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated, treated and lysed in a culture plate. For detection, lysates are subsequently transferred into a 96- or 384-well low volume detection microplate, where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

Product performances

1. Specificity for α-SMA isoform validated by siRNA experiments

The human hepatic stellate cell line LX-2* and the mouse fibroblast cell line NIH/3T3 were plated in culture-treated 96-well plates (10,000 cells/well) in complete culture medium, and incubated for 24h at 37°C - 5% CO2. The day after, the cells were transfected with an Alpha-SMA siRNA or with a negative control siRNA for 24h, and then treated or not with 2.5 ng/mL of TGF-β1 for an additional 24 hours. After medium removal, the cells were lysed with supplemented lysis buffer #3 (50 µL for LX-2 or 200 µL for NIH/3T3) for 30 minutes at RT under gentle shaking. 16 µL of lysate were transferred into a low volume white microplate and 4 µL of the HTRF® Alpha-SMA detection antibodies were added. The HTRF signal was recorded after an overnight incubation at RT.

On both cell lines, transfection with the Alpha-SMA siRNA led to a huge decrease of the HTRF specific signal (~ 60 to 70%) compared to the cells transfected with the negative control siRNA, demonstrating that the HTRF® Alpha-SMA assay is specific for α-SMA isoform and does not cross-react with other actin isoforms.

*Provided by EMD Millipore (product number #SCC064

2. Monitoring of the differentiation of HSCs into myofibroblasts in liver fibrosis

The human hepatic stellate cell line LX-2* was plated in a culture-treated 96-well plate (50,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, cells were treated with increasing concentrations of TGF-β1 for 48 hours in serum-free and antibiotic-free culture medium supplemented with 0.2% BSA. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before adding 4 µL of the HTRF® Alpha-SMA detection antibodies. The HTRF signal was recorded after an overnight incubation.

Image of the validation of the Cisbio HTRF Alpha-SMA on the human hepatic stellate cell line LX-2 treated with TGF-β1

* LX-2 cell line provided by EMD Millipore (product number #SCC064)

TGF-β1 treatment induces a two-fold increase of α-SMA expression level, highlighting the differentiation of HSCs into myofibroblasts. 

3. Monitoring of the differentiation of primary HLFs into myofibroblasts in lung fibrosis

Primary human lung fibroblasts (HLFs) were plated in a culture-treated 96-well plate (20,000 cells/well) in complete culture medium and incubated overnight at 37°C - 5% CO2. Cells were then incubated for an additional 24 hours in serum-free medium before treatment with increasing concentrations of TGF-β1 for 48 hours. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white plate before adding 4 µL of the HTRF® Alpha-SMA detection antibodies.

Image of the validation of the Cisbio HTRF Alpha-SMA on primary human lung fibroblasts treated with TGF-β1

The long-term treatment of cells with TGF-β1 results in a four-fold increase in α-SMA expression level, highlighting the differentiation of HLFs into myofibroblasts. 

4. Analysis of α-SMA expression to monitor the differentiation of fibroblasts into myofibroblasts

The mouse fibroblast cell line NIH/3T3 was plated in a culture-treated 96-well plate (6,250 and 12,500 cells/well) in complete culture medium, and incubated at 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-β1 for 48 hours in serum-free and antibiotic-free culture medium supplemented with 0.2% BSA. After medium removal, the cells were lysed with 200 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the HTRF® Alpha-SMA detection antibodies. The HTRF signal was recorded after an overnight incubation.

TGF-β1 long-term treatment results in a three-fold increase of α-SMA expression level, which demonstrates the differentiation of fibroblasts into myofibroblasts.

5. HTRF Alpha-SMA assay compared to Western Blot

NIH/3T3 cells were seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C - 5% CO2 until 80% confluency was reached. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of the HTRF® Alpha-SMA detection reagents. Equal amounts of lysates were used for a side by side comparison with Western Blot.

Using HTRF® Alpha-SMA detection reagents just 200 cells were sufficient for minimal signal detection, while 800 cells were needed to detect a significant Western Blot signal. Thus the HTRF® Alpha-SMA assay is 4-fold more sensitive than the Western Blot.

Simplified pathway

TGF-β is a profibrotic cytokine that represents the main myofibroblast inducer.

The SMAD-dependent TGF-β signaling pathway involving the activation of the transcription factors SMAD2/3 leads to ACTA2 gene transcription and expression of α-SMA (α-Smooth Muscle Actin). This specific actin isoform is incorporated into actin stress fibers and gives the cells a strong contractile activity characteristic of differentiated myofibroblasts.

TGF-β also signals through SMAD-independent pathways by activating the TAK1-dependent kinases p38, JNK and NF-κB, as well as AKT and ERK kinases.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Alpha-SMA kit - 500 tests62ASMAPEG
Alpha-SMA kit - 10,000 tests62ASMAPEH
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Companion products

DescriptionCat. noProduct insertMSDS
Alpha-SMA kit control lysate62ASMATDA
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