Assay principle

The alpha-SMA assay is based on a TR-FRET sandwich immunoassay format comprising two antibodies, one labelled with Eu3+-Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to Alpha-SMA, and the proximity of donor and acceptor leads to a fluorescent TR-FRET signal.

Assay protocol

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 protocol enables the cells' viability and confluence to be monitored. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. The assay can be run with frozen cell lysates or fresh cells in culture.

Alpha-SMA expression upon differentiation of HSCs into myofibroblasts

​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, the cells were treated with increasing concentrations of TGF-beta1 for 48 hours. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #3, 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. TGF-beta1 treatment induces a two-fold increase of Alpha-SMA expression level, highlighting the differentiation of HSCs into myofibroblasts.​

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

Primary HLFs differentiation 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-beta1 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. The long-term treatment of cells with TGF-beta1 results in a four-fold increase in Alpha-SMA expression level, highlighting the differentiation of HLFs into myofibroblasts.

Alpha-SMA expression upon 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-ß1 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-beta1 long-term treatment results in a three-fold increase of Alpha-SMA expression level, which demonstrates the differentiation of fibroblasts into myofibroblasts.

HTRF Alpha-SMA assay compared to Western Blot

NIH/3T3 cells were seeded in a T175 flask, and incubated for 2 days at 37°C. After medium removal, the cells were lysed 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.

TGF-beta signaling pathway

TGF-beta is a profibrotic cytokine that represents the main myofibroblast inducer. The SMAD-dependent TGF-beta signaling pathway involving the activation of the transcription factors SMAD2/3 leads to ACTA2 gene transcription and expression of alpha-SMA (a-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-beta also signals through SMAD-independent pathways by activating the TAK1-dependent kinases p38, JNK and NF-kappaB, as well as AKT and ERK kinases.