Pe anti human cd133

Primary GBM cells were isolated and incubated with a PE anti-human CD133 (1:50, Biolegend, San Diego, CA) for 30 min at 4°C in dark. Then cells were analyzed by FACS using a FACS CaliburTM flow cytometer (BD Immunocytometry Systems, USA). Dead cells were excluded by propidium iodide (PI) staining. The acquired data were analyzed with FlowJo vX.0.6 software (Tree Star Inc., Ashland, OR).

To delineate cancer stem cell markers CD44 and CD133 expression in HCC cells, flow cytometry was used to detect the levels of the cell surface proteins. Cells were harvested and incubated with FITC mouse anti-human CD44 (BD PharmingenTM, #555478) and PE anti-human CD133 (BioLegend, #372804) in 4 °C for 30 min. The measurement was performed using a FACS Calibur flow cytometer (BD Biosciences).

A549 cells and CSCs were digested with 0.25% trypsin, dispersed into suspension by pipetting, and centrifuged at 1,000 rpm for 5 min to collect the cells. Then the cells were washed twice with PBS, and resuspended in 100 μl PBS (1 × 10⁷ cells/ml). Subsequently, 5 μl PE anti-human CD133 or FITC anti-human CD44 (Biolegend, China) was added to each tube. After 30 min incubation in the dark at 4°C, cells were washed with PBS and resuspended in 200 μl PBS. Finally, cellular fluorescence intensity was analyzed by flow cytometry using FITC channel (excitation: 488 nm and emission: 525nm), PI channel (excitation: 535 nm and emission: 615 nm). To set the background fluorescence levels, PE rat IgG1 κ isotype control and FITC rat IgG1 κ isotype control (Biolegend, China) were used as the negative control.