Anti cd133 apc

Freshly isolated MPCs and P2-MSCs were washed in MACSQuant™ Running Buffer (Miltenyi Biotech, Bergisch Gladbach, Germany) and stained with anti-CD11c VioBlue®, anti-CD18 APC, anti-CD31 PE, anti-CD34 VioBlue®, anti-CD45 APC-Vio770, anti-CD73 PE, anti-CD90 FITC, anti-CD133 APC, anti-CD146 FITC, HLA-DR VioBlue® (Miltenyi Biotech), anti-STRO-1 FITC, and CD144 PE (Biolegend, San Diego, CA, USA). Samples were acquired by MACSQuant® Flow Cytometer and analyzed by MACSQuantify® Software (Miltenyi Biotech).

Mice were euthanized, and brain tumors were dissociated to single cells and stained with anti‐CD133‐APC or anti‐CD15 or anti‐EGFR (Miltenyi) or Annexin V‐PE (BioLegend) and DAPI. Tumor cells were gated based on GFP expression, and mouse cells were gated out using a lineage mixture of Pacific blue‐conjugated H2k^b, H2k^d Ter119, CD31, and CD45 antibodies. Flow cytometric analysis and cell sorting were performed on a BD FACS ARIA II (Becton Dickinson).

The populations of CD34+ and CD133+ endothelial progenitor cells were measured by flow cytometry utilizing a Beckman Coulter Navios (Indianapolis, IN, USA) and were expressed as percent cell count/μL. The following antibodies were used: anti CD45 FITC and anti CD34 PE by Beckman Coulter (Indianapolis IN, USA), anti CD133 APC by Miltenyi Biotec (Bergisch Gladbach, Germany). The software utilized for acquisitions and analyses was Beckman Coulter Navios Software (Indianapolis IND, USA).
The positive population for both CD34 and CD133 antigens has been defined by a Boolean strategy utilizing three dot plots: CD45 vs side scatter to define leucocytes, CD34 vs side scatter to reveal CD34 positive cells and CD133 vs side scatter for CD133 positive cells.

Whole blood staining's were performed within 24 hours after blood collection. White blood cells were counted using Cell-Dyn Sapphire Hematology System (Abbott Diagnostics, Chicago, IL, USA). For CEC and CEP staining 5 millions cells per tube were used, while for monocyte and isotype staining's 1 million cells was used. Directly labeled antibodies were added on total blood and incubated for 20 minutes at 4°C, followed by 10 minutes red-blood-cells lysis (Bühlmann Laboratories, Schönenbuch, Switzerland) and washing using cold PBS.
All anti-human antibodies were used at the concentration give in manufacturer's instructions of use: anti-CD45-V450, anti-CD146-Pe, anti-CD31-AlexaFluo 647, anti-CD34-PeCy7, anti-VEGFR2-Pe, anti-CD11b-V450, anti-CD64-FITC, anti-JAM1-Pe, anti-TIE2-AlexaFluo 647, anti-KIT-PeCy7, anti-CD195-Pe, mouse IgG2a-AlexaFluor 647, mouse IgG1-Pe, mouse IgG1-PeCy7, mouse IgG1-APC, mouse IgG2a-Pe, 7AAD (all from Becton Dickinson), Syto16 (Life Technologies, Carlsbad, CA, USA), anti-CD133-APC (Miltenyi), anti-VEGFR1-APC (R & D Biosystems, Minneapolis, MN, USA).
BD FACSCanto II (Becton Dickinson) instrument was used to perform experiments and FlowJo 9.8.3 (Treestar Inc., Ashland, OR, USA) software was used to analyze all data.

For flow-cytometric analysis hematopoietic cells and ECs were stained with different combinations of monoclonal fluorochrome-conjugated antibodies (see Supplemental Table 5) for at least 20 min at 4 °C. Propidium-Iodide (PI) or 7-Aminoactinomycin D (7-AAD) were used for dead cell exclusion. Appropriate isotype-matched, control monoclonal antibodies were used to determine the level of background staining in all experiments. Flow cytometric analyses were performed on a FC500 flow cytometer equipped with the CXP 2.2 software (Beckman Coulter) (Fig. 1C). Cells were sorted using a FACSAria I cell sorter. The sort-purity was routinely assessed by recovery of sorted cells and was >99.5%.
Samples from murine BM were stained with the following fluorochrome conjugated antibodies: anti-CD34-APC-AF750 (Beckman Coulter), anti-CD133-APC (Miltenyi Biotec), anti-CD45RA-BV711 (BioLegend), anti-CD38-BV785 (BD Biosciences), anti-CD10-PeCF594 (BD Biosciences) and CD45-BV510 (BD Biosciences). DAPI staining was used for excluding dead cells. All measurements were performed in a BD FACSAria^TMIII (Beckman Coulter) flow cytometer.

To detect the expression of cell surface markers, MIHA, Hep3B, and Huh7 cells were seeded in 60 mm dishes at 3 × 10⁵ cells/dish, incubated for 48 h, and then harvested. The cells were washed and stained with anti-EpCAM-FITC (1:50, BD Biosciences, San Jose, CA, USA) and anti-CD133-APC (1:50, Miltenyi, Bergisch Gladbach, Germany) antibodies for 10 min in the dark at 4 °C according to the manufacturer’s protocol. Unstained cells served as the gating control for each cell line.
For cell death analysis, untreated, CAP-exposed, or PAM-treated, MIHA, Hep3B, and Huh7 cells were harvested 72 h after the initial treatment. The cells were washed with phosphate buffer saline (PBS), resuspended in 1X binding buffer, followed by incubation with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI; BD Biosciences) for 15 min according to the manufacturer’s protocol.
After staining, 10,000 cells were analyzed per assay using a FACSCalibur (BD Biosciences) flow cytometer and FlowJo V10 software.