Flow cytometry-based analysis of CD133 and CD44 expression and magnetic sorting-based separation

After lung cancer cells were cultured overnight, the cells were trypsinized, washed and suspended in PBS. The cells were then incubated with fluorescent antibody (phycoerythrin-labeled CD133 antibodies; cat. no. FAB11331P-025; and Alexa Fluor^® 488-labeled CD44 antibodies; cat. no. FAB6127G; R&D Systems, Inc.) at a final concentration of 1 µg/ml on ice in a refrigerator. After 1 h, the cells were washed with PBS to eliminate any unbound fluorescent antibody. Finally, the washed cells were suspended in PBS for immediate analysis by fluorescence-activated cell sorting (FACS) using a FACSCalibur (BD Biosciences). CD133⁺ or CD44⁺ cells were separated using a magnetic column included in the MicroBead kit according to the manufacturer's protocol [CD133 MicroBeadkit (cat. no. 130-100-857) and CD44 MicroBeadkits (cat. no. 130-095-194); both Miltenyi Biotec). The cells were centrifuged and the supernatant was removed. Beads were added and incubated with the cells. Prior to sorting, the column was placed in a magnetic field and rinsed, and the cells were then loaded onto the column. The column was then added to another tube and marker-negative cells were collected. Finally, the proportion of positively-stained cells was analyzed as described above. The rat IgG2B Alexa Fluor^® 488-conjugated (cat. no. MAB0061; R&D Systems, Inc.) or phycoerythrin-labeled isotype (cat. no. IC013P; R&D Systems, Inc.) control antibodies with a dilution of 1:500 were used as the negative controls.