Cell proliferation assay

The protocol for cell proliferation assay under compression

1) Plate 1 mL of MDA-MB-231 cells with the density of 2×10⁵ cells/mL into transwell insert (ThermoFisher, #140640, 0.4 μm pore);

2) Add 2 mL of media into the well of 6 well plate;

3) After culturing for 24 h at 37 °C , 5% CO₂, put agarose gel (make sure that the gel is plate in both of two sides, pre-incubated for 1 h at 37 °C to warm the gel) above the cells.

4) Putting different weights on the gels;

5) After culturing for 12 h, carefully take out the weight and agarose gel in the insert and the media in the bottom well;

6) Aspirate the media in the different inserts into different centrifuge tubes;

7) Adjust their volume to 1 mL;

8) Add back these media to the original corresponding insert.

9) Add 100 μL WST-8 mixture to the insert and incubated at 37 °C, 5% CO₂ for 2 h

10) Mix gently on an orbital shaker for one minute to ensure homogeneous distribution of color.

11) Aspirate 100 μL reaction suspension to 96 well plate (8 wells for each group)

12) Measure the absorbance of each sample using a microplate reader at a wavelength of 450 nm.