Calcein am fluorescent probe

Stromal cells (1 × 10⁵ cells/ml) were plated on a 24-well plate and cultured for 24 h. In accordance with the instructions of the manufacturer, Nalm-6/RS4;11 cells were stained with Calcein-AM fluorescent probe (Beyotime Biotechnology Co., Ltd., Shanghai, China) for 30 min to label as green fluorescence, then washed with PBS, and resuspended in a fresh medium to make the cell density 4 × 10⁵/ml. Subsequently, 500 μl leukemia cells suspension was added to each 24-well plate of stromal cells, the suspended supernatant was gently aspirated after co-culturing for 24 h, and the adhered leukemia cells labeled with green fluorescence were checked under a fluorescent microscope. For quantitative analysis in each group, 5 random fields of view were taken for the adhered cell count, and then the adhesion rate of leukemia cells was calculated by dividing the number of adhered cells of the experimental group by the number of adhered cells of the control group, and the values of control group were normalized to 100%.

After treatment with Yap siRNA or its inhibitor Verteporfin in the existence or not of GSH, stromal cells were incubated with Calcein AM fluorescent probe (Beyotime, #C2009S) concomitant with the supplementation of fluorescence quenching solution for 25 min at 37 °C. Finally, cells were analyzed by flow cytometry to determine the opening of mPTP.