Cells were cultivated in standard culture medium for 3 days. CD133-PE antibody (Miltenyi Biotec GmbH, Germany) was used at a 1:50 dilution and incubated for 15 min with 500.000 tumor cells per sample. Dead cells were excluded based on DAPI staining. Cells were analyzed using BD Canto II cytometer and FCS Express software was used for the evaluation.
Cd133 pe antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
The CD133-PE antibody is a laboratory tool used for the detection and analysis of CD133-positive cells. CD133, also known as prominin-1, is a cell surface glycoprotein that is expressed on various stem and progenitor cells. The CD133-PE antibody is conjugated with the fluorescent dye phycoerythrin (PE), allowing for the identification and quantification of CD133-positive cells through flow cytometry or other analytical techniques.
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Lab products found in correlation
Fcr blocking reagent, by Miltenyi Biotec (3 mentions)
Facsaria 3, by BD (2 mentions)
Canto 2 cytometer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facsaria system, by BD (1 mentions)
Cflow plus software, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Easysep human pe positive selection kit, by STEMCELL (1 mentions)
Annexin 5 pe, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Cd44 microbeads, by Miltenyi Biotec (1 mentions)
Anti pe multisortmicrobeads, by Miltenyi Biotec (1 mentions)
Compensation beads, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti human igg fc, by BioLegend (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
14 protocols using cd133 pe antibody
1
CD133 Expression in Tumor Cells
2
Flow Cytometry Analysis of Cell Surface Markers
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Trypsinization was performed to harvest transfected cells. Cells (105) were incubated with phycoerythrin (PE)-conjugated immunoglobulin G (IgG) 1 (cat. no. 130-112-760; Miltenyi Biotec GmbH) or CD133-PE antibody (cat. no. 130-093-193; Miltenyi Biotec GmbH) at 4°C for 15 min. Cells were then resuspended in PBS and signals were detected using FACS Aria system (BD Immunocytometry Systems) and processed by CellQuest software, version 5.1 (Becton, Dickinson and Company).
3
Isolation and Identification of CSCs
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The suspended MDA-MB-231, MCF-7, and TA2 cells were fixed in 75% cold ethanol, and 106 cells were incubated with CD133-PE antibody (1:11 dilution, Miltenyi Biotec) solution or isotype control on ice for 15 min before they were washed and resuspended in staining buffer (2% fetal calf serum in PBS). ALDH1+ cells were detected and isolated with the ALDEFLUORTM stem cell isolation kit (STEMCELL Technologies). 1 mL of the suspended tumor cells was added into the test tubes, and 5 μL ALDEFLUORTM DEAB was added into the control tubes. Activated ALDEFLUOR at a 5 μL volume was added into the test tubes, and half of the mixture in the test tubes was then removed into the control tubes. All tubes were incubated at 37°C for 30 min. Approximately 0.5 mL ALDEFLUOR assay buffer was added to resuspend the cells. All cells were analyzed and isolated using FACSAria III (BD Biosciences). Gates were set with isotype controls such that, for each cell, less than 1% of the total cell population was false-positive. The labeled cells were then analyzed (10,000 events), and the data were saved as FACS.
4
CD133 Expression Analysis of H460 Cells
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H460 were seeded in low attachment plates (Corning) with the concentration of 10,000 cells/ml. Cells were cultured for 5 days, then digested and collected. The cells were re-suspended in PBS at 2 × 106/100 ul. Unstained cells, isotype and single color-stained cells were used for controls. CD133-PE antibody (Miltenyi, Biotec) was used for CD133 staining. The results were examined with BD Accuri™ C6 flow cytometer (Becton-Dickinson, CA, U.S.) and analyzed with CFlow Plus software (Becton-Dickinson, CA, U.S.).
5
Isolation and Characterization of CD133+ Cancer Cells
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Human colon cancer cell lines HT29, HCT-116, and SW620 were dissociated into single-cell suspensions, washed with phosphate-buffered saline (PBS), and stained with an AC133/CD133-PE antibody (Miltenyi Biotec). Magnetic cell separation was performed on a column-free cell isolation platform using an AC133/CD133-PE antibody (Miltenyi Biotec) and the EasySep™ Human PE Positive Selection Kit (STEMCELL Technology, USA). After magnetic sorting, both the CD133+ and CD133- cell populations were assessed by flow cytometry using a CD133-PE antibody (Miltenyi Biotec). Apoptosis was measured by labeling cells with 7-AAD-PE and Annexin V-PE (BD Biosciences, CA), as recommended by the manufacturer.
6
Flow Cytometric CD133+ Cell Quantification
7
Identification of CD133 Cancer Stem Cells
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To identify the cancer stem cell marker CD133, cells and spheres were dissociated as a single-cell suspension and washed with PBS. The cells were labeled with the CD133-PE antibody (Miltenyi Biotec, San Diego, CA, USA) supplemented with FcR Blocking Reagent (Miltenyi Biotec) and analyzed using a FACS LSRII flow cytometer.
8
Isolation of Cancer Stem Cell Subpopulations
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Cells positive for CD44 and CD133 surface markers were isolated from cultured Hep2 and TU-177 cells by MACS by using CD44 MicroBeads or CD133-PE antibody (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. Briefly, 107 cells in 100 μL buffer were incubated with 10 μL CD133-PE antibody. Cells were washed with 1 mL buffer and centrifuged at 300×g for 10 min, then the cell pellet was resuspended with 90 μL buffer, and 10 μL anti-PE Multisort MicroBeads (Miltenyi Biotec) was added and incubated for 15 min in the dark at 4℃. Cells were washed and underwent magnetic sorting, then CD133-enriched (CD133+) and CD133-depleted (CD133-) cell populations were collected. For 107 cells of CD133+ or CD133-, 20 μL CD44 MicroBeads was added and incubated for 15 min in the dark at 4 ℃, followed by washing and magnetic sorting, then CD133+CD44+, CD133+CD44-, CD133-CD44+, and CD133-CD44- cells were obtained. After isolation, cells with the 4 types of surface markers were cultured for further analysis, and some of these cells were used to evaluate the efficiency of magnetic separation by flow cytometry (Becton Dickinson).
9
Isolation of CD133+ Cancer Stem Cells
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The NSCLC cell lines (A549, H460 and H2170) were harvested by 5-min incubation with 0.25% trypsin–EDTA followed by centrifugation. The cell pellets were then suspended in DPBS with 2% FBS and transferred into 75-mm polystyrene round-bottom test tubes. CD133-PE antibody, 1:10 dilution (Clone: AC133; Isotype: Mouse IgG1 kappa) (Miltenyi Biotec, Bergisch Gladbach, Germany), was added to the cell suspension and incubated for 15 min in dark. Cells were then washed with DPBS containing 2% FBS and filtered through a 40-μM cell strainer, before being subjected to specific CD133+ (cancer stem cells, CSCs) and CD133− (non-CSCs) isolation using a fluorescence-activated cell sorter (FACSAria III; BD biosciences, San Jose, CA, USA). The isolated CD133+ CSCs were grown in complete RPMI-1640 medium and supplemented according to cell types (described as complete medium for H460, H2170, and A549), and re-sorted for another two cycles for a total of three sorting experiments to get a pure CD133+ population.
10
CyTOF Analysis of Stem Cell Markers
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