Pe anti mouse cd133

The cultured cells were dissociated by TrypLE (Gibco) into a single cell suspension, and then fixed with 4 % paraformaldehyde and permeabilized by ice-cold methanol. Before staining, the sample cells were washed and resuspended at 5 ~ 10 × 10⁶ cells/ml in PBS containing 1 % BSA and 0.03 % NaN₃ (wash buffer) and then stained by the specific antibodies (PerCP-Cy™5.5 Mouse anti-Sox2, BD Biosciences; Alexa Fluor® 647 Mouse anti-GFAP, BD Biosciences; Alexa Fluor® 647 Mouse anti-Nestin, BD Biosciences; Alexa Fluor® 488 Mouse anti-Ki-67, BD Biosciences; PE anti-mouse CD133, Biolegend, San Diego, CA, USA). After antibody conjugate was added, the cells were incubated in the dark on ice for 30 min, and washed twice with wash buffer. The cell samples were resuspended and analyzed on a flow cytometer (Accuri C6, with the C6 software, BD Biosciences).

After seeded in confocal dish (2 × 10⁴ cells per well) for 24 h, B16F10 tumorsphere cells were incubated with hMnO₂@gCMs for another 2, 4 or 8 h, the hMnO₂@gCMs was stained with DiD in advance. After being stained with DAPI and phalloidin-FITC, the cells were observed under a confocal laser scanning microscopy (CLSM, ZEISS LSM980). To determine the cellular uptake by flow cytometry (FACS), the tumorsphere cells were incubated overnight and then incubated with hMnO₂@gCMs labeled with DiO for 2, 4 or 8 h. After staining with PE anti-mouse CD133 (Biolegend, 141203), the cells were detected by FACS. For lysosomal escape analysis, 24 h after seeding in confocal dish (2 × 10⁴ cells per well) for, CSCs were incubated with DIO-labeled hMnO₂@gCMs for additional 1, 2, 4 or 6 h. After being stained with Lysotracker Red, the cells were observed under CLSM.