In vitro cytotoxicity assay

1.     Seed 1 x 10⁴ MDA-MB-231 cells in a 96-well plate and incubate at 37 °C for 12 hours.

2.     Wash the cells with PBS for 1 time.

3.     Dilute free cabazitaxel or nanocomplexes in DMEM medium containing 10% FBS.

4.     Add the diluted solutions into each well (100 µL/well) and incubate at 37 °C for 48 hours.

5.     Add Triton X-100 (5 µL in 100 µL DMEM medium containing 10% FBS) to untreated cells as a positive control.

6.     After 48 hours, add 10 µL of 3-(4,5-dimethyltjiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) to each well.

7.     Incubate the plate at 37 °C for 2 hours.

8.     Carefully remove the medium. (Do not disturb the cells and do not rinse with PBS).

9.     Add 200 µL DMSO per well.

10.  Cover the plate with tinfoil and agitate cells on an orbital shaker for 5 min slowly.

11.  Add 25 µL Sorensen’s glycine buffer per well and shaker slowly for 10 min.

12.  Read the absorbance at a wavelength of 570 nm with a reference wavelength of 650 nm.

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