Fibroblast contraction assay
Reagents:
- Cell Contraction Assay Kit from Cell Bio Labs Inc., Cat: CBA201.
- NIH 3T3 fibroblast.
- Recombinant TGF-beta from R and D systems, Cat: 240-B.
The process of fibrosis in cardiomyopathy depends on the transition of fibroblast to myofibroblast. Fibroblast contractivity is the ultimate approach to assess myofibroblast formation. We use TGF-beta to induce myofibroblast formation using the following protocol and identify molecules which may control the transition.
1. Seed NIH 3T3 fibroblast in 6-well plate, 600, 000 cells per well.
2. Next day, treat cells with recombinant TGF-beta for 24 hrs.
3. Next day, transfect plamids of your favourite molecules to assess effects on myofibroblast contraction.
4. Next day, harvest cells for collagen gel culture in 24 well plates.
5. For each well, we use 100 ul cell suspension of around 2-5 million cells and 500 ul collagen gel mix.
6. Prepare collagen gel mix by the following regime for each well (all provided in the Assay kit). Keep all the solutions on ice. In a cold sterile tube, add collagen solution, 5 X DMEM, mix well. Then add neutralization solution, mix it immediately and keep the gel mix on ice.
- Collagen solution: 400 ul
- 5 X DMEM: 103 ul
- Neutralization solution: 13.5 ul
7. In labelled cold sterile tubes, add 100 μl of cells and 500 μl of collagen gel mix. Mix well while still keeping them on ice.
8. Add the cell-collagen mix into the 24 well plate. Avoid bubbles. Swirl gently to cover the complete surface of well.
9. Incubate in 37 C for 1 hrs.
10. Detach the gel from the plate with a sterile blade or needle.
The Gel should be free floating else it won’t contract.
11. Add 1 ml culture media (DMEM - 10% FCS) at the top of the gel very gently.
12. Take picture for 0 hour. Then as often as required.
Courtesy: Bajaj N.