Cd133 pe

The EPCs were identified by flow cytometry [18 (link),19 (link)]. Cells were washed twice with cold PBS and incubated with rabbit PE-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany) and AF647-KDR (Becton Dickinson, Franklin Lakes, NJ, USA) for 30 min at room temperature. The normal rabbit IgG labeled by fluorescein was used as a control to define the negative population for each stain. CD31 (Becton Dickinson, Franklin Lakes, NJ, USA) and CD34 (Becton Dickinson, Franklin Lakes, NJ, USA) were not directly labeled and needed to be incubated with labeled secondary antibodies before flow cytometry. Cells were analyzed by Calibur (BD).
For the testing of EPC number, the CD34-positive population of mononuclear cells was isolated from the peripheral blood cell population, and then CD45dim cells were selected from the CD34-positive population. CD34/CD45dim cells were further sub-gated to identify the KDR-positive cell population, and the CD133-positive cell population was selected from the KDR-positive cell population.

Cells that reached 80% confluency were exposed to a single dose of 5 Gy irradiation and were collected after further culture for 15–16 h and the pellets were rinsed with washing buffer (PBS containing 0.5% BSA). Cell pellets were resuspended in pre-diluted FITC mouse anti-human CD47 antibody (BD Pharmingen Catalog #556045) and incubated in room temperature for 1 h in dark, followed by three times washing with washing buffer. For detection of the co-expression of stem cell markers on RR U87 and RR U251, the double staining of PE-CD133 (Miltenyi Biotec, Catalog #130-080-801), and APC-HER2 (R&D Catalog #FAB1129A) were performed. All antibodies were titrated to optimized conditions. The FACS Canto II cytometer (BD) and FlowJo (Three Star, Ashland, OR, USA) were used for analysis. The gating strategies were based on FSC and SSC. The appropriate channels of cell populations, such as unstained and positive control, were gated as standard.

Flow cytometry was used
to isolate the CD133⁺/CD44⁺ cells in the HT-29
cell line. A total of 1 × 10⁶ cells/mL were labeled
by the incubation with 10 μL of CD133-PE and CD44-APC (Miltenyi
Biotec, Bergisch Gladbach) for 10 min at 4 °C. The CD133⁺/CD44⁺ subpopulation was then filtered and isolated
by a FACSAria II flow cytometer for CD133⁺/CD44⁺ markers to gain HT-29 CSCs.^{31 (link)} HT-29 CSCs
were grown in HT-29 cell culture conditions.

To analyze the apoptotic nature of the putative A549 stem cells, the Annexin V-PE Apoptosis Detection Kit I (559763; BD Biosciences, San Jose, CA, USA) was used. A total of 5 μL Annexin V-PE and 5 μL 7-AAD in 100 μL binding buffer were incubated with 1×10⁶ cells for 15 min at room temperature in the dark. Then the cells were washed twice with phosphate-buffered saline (PBS) and resuspended with 400 μL binding buffer. Flow cytometry was used for detection (Accuri C6, BD Biosciences). To analyze the relative CD133, ABCG2, OCT4, and SOX2 gene expression in the putative A549 stem cells, the cells were incubated with 100 μL PBS containing 5 μL CD133-PE (Miltenyi Biotec, Teterow, Germany) and 2.5 μL ABCG2-PE (Miltenyi Biotec) antibodies for 30 min in the dark. Then the cells were permeabilized in cold methanol and incubated with 2.5 μL OCT4-PE (eBioscience, San Diego, CA, USA) and 2.5 μL SOX2-FITC (eBioscience) antibodies for 30 min in the dark. The cells were washed twice with PBS and resuspended with 200 μL PBS for measurement by flow cytometry.

The CD133⁺ cells were enriched by the magnetic
activated cell sorting (MACS) method (Miltenyi Biotec,
Canada) according to the manufacturer’s instructions.
Repeating the procedure resulted in higher purity of the
selected CD133⁺ cells. The efficiency of purification was
verified by flow cytometry (Partec PAS III, Germany),
with cells counterstained with the monoclonal antibodies
(moAb) CD133-PE and CD34-FITC (Miltenyi, Canada)
since most CD133+ cells also express CD34. In addition,
moAb CD41-PE and CD61-FITC (DAKO, Denmark)
were used to confirm the negativity of the megakaryocyte
cells within separated cells.