Alexa488 alexa594 conjugated secondary antibody

One million trypsinized cells were incubated with an anti-CD133 antibody for 45 min. After incubation, the cells were washed and then incubated with an Alexa488/Alexa594-conjugated secondary antibody (Invitrogen) for 30 min and washed again before analysis using a BD FACScaliber flow cytometer (BD Biosciences, San Jose, CA, USA). The fluorescent intensities were analyzed with Cell Quest Software (BD Biosciences). The antibodies conditions are listed in Table S1.

H441 and A549 cell lines were washed with 1 × PBS and harvested prior to antibody incubation. Flow cytometry technique was used to examine CD133 expression on single cells detached from plate. For 45 min, one million trypsinized cells were treated with an anti-CD133 antibody (Cell Signaling Technology Inc., Beverly, MA, USA) or an isotype control IgG (Upstate Biotechnology, Lake Placid, NY, USA). Following washing, the cells were incubated for 30 min with an Alexa488/Alexa594-conjugated secondary antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) before being analyzed with a BD FACScaliber flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cell Quest Software was used to examine the fluorescent intensity (BD Biosciences, Franklin Lakes, NJ, USA).