Canto 2 analytical cytometer

A previously trypsinized suspension of 10⁶ cells was resuspended in 125 µl of PBS containing 2% FBS and 5 mM EDTA. This suspension was then blocked by adding 12.5 µl of blocking agent (Miltenyi Biotec) and incubated for 10 min on ice. An anti-CD133 antibody conjugated to the fluorochrome phycoerythrin (PE) (MACS) was then added at a 1:25 dilution and incubated for 30 min on ice in the dark. A tube containing 1–106 cells was left unlabeled with the antibody to use as a negative control for staining. After the end of the incubation period, the cells were washed 2 times with PBS containing 2% FBS and 5 mM EDTA. Subsequently, the cells were centrifuged for 5 min at a speed of 1000 rpm (112 g) and resuspended in 500 µl of PBS containing 2% FBS and 5 mM EDTA. Finally, the cell suspension was examined on a Canto II analytical cytometer (BD Biosciences), with the cells separated into positive and negative populations based on staining for the CD133 marker in certain cases.

The previously trypsinized suspension of 1 × 10e6 cells was resuspended in 125 µl of phosphate-buffered saline (PBS) with 2% FBS and 5 mM EDTA. This suspension was then blocked by adding 12.5 µl of blocking agent (Miltenyi Biotec) and incubated for 10 min on ice. The anti-CD133 antibody conjugated to fluorochrome phycoerythrin (MACS) was then added at a 1 : 25 dilution and incubated for 30 min in ice and darkness. A tube with 1 × 10e6 cells was left unlabeled with the antibody to use as a negative control for staining. After the end of the incubation period, the cells were washed two times with PBS, with 2% FBS and 5 mM EDTA. Subsequently, the cells were centrifuged for 5 min at a speed of 1000 r.p.m. and resuspended in 500 µl of PBS with 2% FBS and 5 mM EDTA. Finally, the cell suspension was examined in a Canto II analytical cytometer (BD Biosciences), separating in certain cases two populations positive and negative for the CD133 marker.