Qcm 24 well colorimetric cell invasion assay kit

Caco-2 cell invasion was assessed using a QCM™ 24-well Colorimetric Cell Invasion Assay kit (Chemicon International, Temecula, CA, USA (Cat # ECM551)), according to the manufacturer’s instructions. Cells were starved of serum for 24 h. Then, cells were collected, suspended in serum-free Dulbecco’s Modified Eagle Medium (DMEM) growth media containing either IC₅₀ dose of retinoids or 0.1% DMSO (negative control), and seeded into the upper chamber of inserts at a density of 2.5 × 10⁵ cells per insert and divided into two groups for either 24 or 48 h. Inserts were further added into a 24-well plate containing 500 µL of 10% FBS-containing DMEM per well. After incubation for 24 or 48 h at 37 °C, remaining media was removed from the invasion chamber, and cells that had migrated to the other side of the membrane were stained with “Cell Stain” solution for 20 min at room temperature. Non-invaded cells were carefully removed from the upper side of the chamber with the help of cotton-tipped swabs. The stain of invaded cells was then extracted by 200 µL of extraction buffer in a clean well for 15 min at room temperature. Finally, 100 µL extracted stained solution was transferred into a 96-well plate and the optical density was measured at 490 nm with BioTek 800 TS microplate reader.

Cell proliferation kit (XTT) (Roche, Indianapolis, IN, USA) was used to measure cell viability according to the manufacturer's manual [50 (link)]. Focus formation assay was performed as a more long-term proliferation assay and soft agar assay was performed to detect cell anchorage-independent growth ability. Wound healing assay and a Cell Migration Assay kit (MILLIPORE, Cat. No. ECM508) were used to detect cell migration ability. Quantification of cell invasion was performed using QCM™ 24-Well Colorimetric Cell Invasion Assay Kit (Chemicon International, Temecula, CA, USA). All experiments were done in triplicates and results were presented as the mean ± SD.