AlphaLISA Human and Mouse α-SMA Detection Kit, 5,000 Assay Points

Protocol of the AlphaLISA human & mouse α-SMA assay

The AlphaLISA α-SMA assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples (cell lysates) or control lysate are dispensed directly into the assay plate for the detection of α-SMA by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.

Selectivity for α-SMA isoform validated by siRNA experiments

The mouse fibroblast cell line NIH-3T3 was plated in culture-treated 96-well plates (10,000 cells/well) and incubated for 24h at 37°C - 5% CO₂. The day after, the cells were transfected with an α-SMA siRNA or with a negative control siRNA for 24h and then treated or not with 5 ng/mL of TGF-ß1 for an additional 24 hours. After medium removal, the cells were lysed with 50 µL of lysis buffer for 30 minutes at RT under gentle shaking.

5 µL of lysate were transferred into a 384-well microplate before the sequential addition of AlphaLISA α-SMA detection reagents. Transfection with the α-SMA siRNA led to a huge decrease of the AlphaLISA specific signal (~ 90%) compared to the cells transfected with the negative control, demonstrating that the AlphaLISA α-SMA assay is specific for α-SMA.

MRC-5 differentiation into myofibroblasts in lung fibrosis

Human lung fibroblasts MRC-5 cells were plated in a culture-treated 96-well plate (25,000 cells/well) in complete culture medium. Once adhered, the supernatant was discarded and the cells were stimulated with increasing concentrations of TGF-ß in serum-free culture medium supplemented with 0.1% BSA for 24h at 37°C - 5% CO₂.

After medium removal, cells were lysed with 50 µL of lysis buffer 1X for 30 minutes at RT under gentle shaking, and 5 µL of lysate were transferred into a 384-well plate before adding AlphaLISA α-SMA detection reagents. The treatment of cells with TGF-ß results in an 8.5-fold increase in α-SMA expression level, highlighting the differentiation of MRC-5 into myofibroblasts.

α-SMA expression upon differentiation of fibroblasts into myofibroblasts

The mouse fibroblast cell line NIH-3T3 was plated in a culture-treated 96-well plate (20,000 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-ß for 48 hours in serum-free culture medium supplemented with 0.1% BSA. After medium removal, the cells were lysed with 200 µL of lysis buffer 1X for 30 minutes at RT under gentle shaking, and 5 µL of lysate were transferred into a 384-well plate before the addition of AlphaLISA α-SMA detection reagents.

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

Detection of α-SMA expression into cancer cell line

The Human cancer cell line HepG2 was plated in a culture-treated 96-well plate (from 10,000 to 40,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO₂ for 24h.

The day after, the supernatant was removed, and the cells were lysed with 50 µL of lysis buffer 1X for 30 minutes at RT under gentle shaking. For detection, 5 µL of lysate were transferred into a 384-well plate before the addition of AlphaLISA α-SMA detection reagents. As expected, α-SMA is well detected proportionally to the cellular densities in this oncological model.