Cell Counting Kit-8

Protocol tips

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	- Plates are incubated for an appropriate length of time (e.g. 0, 24, 48, or 72 hours) in the incubator. - Cells were incubated for 1.5 h at 37 °C after addition of CCK-8 reagent

Protocol tips
- Plates are incubated for an appropriate length of time (e.g. 0, 24, 48, or 72 hours) in the incubator. - Cells were incubated for 1.5 h at 37 °C after addition of CCK-8 reagent

Publication protocol

Cell viability was detected using CCK-8 assay (Dojindo, Kumamoto, Japan). Transfected cells were seeded in 96-well plates at 4 × 103 cells/well, and cultured for 0, 24, 48, or 72 h. Then, 10 μL of CCK-8 reagent was added to 100 μL of fresh medium per well. Cells were incubated for 1.5 h at 37 °C. Cell viability was assessed at an absorbance of 450 nm. For the wound healing assay, 1 × 104 cells were added into six well plates and grown to 50–70% confluence. Then cells were scratched by a 1-mL pipette tip and cultured for 48 h. Wound healing areas were examined under inverted microscope. We used Image J software to measure the wound area. The wound closure rate = (Area of original wound − Area of actual wound at 48 h)/Area of original wound × 100%. For invasion assays, transwell vessels (Corning, NY, USA) with 8-μm pores were used. We added 100 μL RPMI-1640 medium without serum containing 1 × 103 cells into the upper chamber precoated with Matrigel (BD Bioscience, Mississauga, ON, Canada). Then, RPMI-1640 medium with 10% fetal bovine serum (FBS) was added into the lower chamber. After 48 h incubation, non-invaded cells were carefully wiped out with a cotton bud. Next, the membrane with cells were fixed by 4% paraformaldehyde and stained by 0.2% crystal violet. Five high power fields (200×) were randomly selected and cells were counted.

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