Mouse cd133 pe antibody

A total of 1 × 10⁷ cells was harvested and suspended in 100 μL of buffer. Then, 10 μL mouse CD133‐PE antibody (Miltenyi Biotec, Teterow, Germany) was added and incubated in the dark at 4°C for 10 minutes. The cells were washed twice with buffer and suspended in 500 μL of buffer for analysis by flow cytometry (BD FACSCalibur, San Jose, California, USA). A mouse IgG1 isotype antibody (Miltenyi Biotec) was used as the control. This experiment was repeated three times. We used the CD133 MicroBead Kit (Miltenyi Biotec) for cell sorting. A total of 1 × 10⁷ cells was harvested and suspended in 60 μL of buffer prior to the addition of 20 μL FcR blocking reagent and 20 μL CD133 MicroBeads, and the mixture was incubated for 10 minutes at 4°C. The cells were washed twice with buffer and then suspended in 500 μL of buffer. Next, magnetic separation was performed according to the manufacturer's instructions. Unlabeled cells passed through, while labeled cells were retained in the column. Labeled and unlabeled cells were separately collected for further experiments.

LO2, HepG2, and SMMC‐7721 were obtained from the American Type Culture Collection (ATCC), maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and antibiotics (1% penicillin and 100 μg/mL streptomycin; Beyotime), with an atmosphere containing 5% CO2 at 37°C. JC‐1 (T4069) and 3‐MA (M9281) were from Sigma‐Aldrich, Puerarin 6″‐O‐xyloside was purchased from Tauto Biotech (E‐1160, Shanghai, China), and Cell Counting Kit 8 (CCK‐8, C0037), PBS, Crystal Violet Staining Solution (C0121), 4% Paraformaldehyde Fix Solution, Colorimetric TUNEL Apoptosis Assay Kit and Bicinchoninic acid (BCA) protein assay reagent (P0012S) were purchased from Beyotime. Mouse CD133‐PE antibody was purchased from Miltenyi Biotec; antibodies of Ki67 (ab15580), Cleaved Caspase‐3 (ab32042), Caspase‐3 (ab90437), OCT4 (ab109183), LC3B (ab229327), Beclin1 (ab207612), ATG7 (ab133528), AKT (ab18785), p‐AKT (ab8933), mTOR (ab32028), and p‐mTOR (abs) were purchased from Abcam. PI3K p110α (#4255), PI3K p85 (#4257), p‐PI3K p85 (#4228), GAPDH (#5174) antibodies and all secondary antibodies were purchased from CST (Danvers, MA, USA). Annexin V fluorescein isothiocyanate/propidium iodide (FITC/PI) kit was purchased from BD Biosciences. Other reagents were purchased from Beyotime and Solarbio.