The cells were dissociated into single cells and labeled with PE-conjugated anti-CD24 and FITC-conjugated anti-CD133 (BD PharMingen) at 4 °C for 20 min. Concentrations of antibodies were used according to the manufacturers’ recommendations. The stained cells were analyzed with the FACS Calibur machine and Cell Quest software (BD Biosciences).
Pe conjugated anti cd24
Manufactured by BD
Sourced in United States
PE-conjugated anti-CD24 is a fluorescently labeled monoclonal antibody that binds to the CD24 cell surface antigen. CD24 is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed on various cell types. This product can be used for the detection and analysis of CD24-expressing cells by flow cytometry.
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Lab products found in correlation
Apc conjugated anti cd44, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Fitc conjugated anti cd133, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur machine, by BD (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Pe conjugated mouse igg, by BD (1 mentions)
Anti e cadherin, by Agilent Technologies (1 mentions)
Anti bmi1, by Abcam (1 mentions)
Anti erα, by Santa Cruz Biotechnology (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti total erk1 2, by Cell Signaling Technology (1 mentions)
Anti mek1 2, by Cell Signaling Technology (1 mentions)
Fluorescein isothiocyanate fitc conjugated anti cd44, by BD (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Anti h3k4me3, by Cell Signaling Technology (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti setd1a, by Fortis Life Sciences (1 mentions)
9 protocols using pe conjugated anti cd24
1
Characterization of CD24/CD133 Subpopulations
2
Characterization of Breast Cancer Cell Lines
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BT474, T47D, MDA-MB-231, BT549 and SKBR3 breast cancer cell lines were purchased from the Committee on Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The pEGFP-C1-ERα plasmid was purchased from Addgene (Cambridge, MA, USA), and pcDNA3-BMI1 plasmid was a gift from Dr. MH Yang (Institute of Clinical Medicine, National Yang-Ming University, Taipei city, Taiwan) [20 (link)]. A wild type BMI1 promoter (pXP2-BMI1-Luc1000) from −1023 to −1 was constructed and a mutant BMI1 promoter was generated by replacing a TGACC (−178–174) sequence in the wild type BMI1 promoter with a GACCC sequence. The following antibodies were used in this study: anti-E-cadherin (DAKO, Glostrup, Denmark; NCH-38), anti-Bmi1 (Abcam, Cambridge, UK; ab126783), anti-ERα (Santa Cruz Biotechnology, CA, USA; sc-543), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA; sc-4777), PE-conjugated anti-CD24 (BD Biosciences, Bedford, MA, USA; ML5), APC-conjugated anti-CD44 (BD Pharmigen, San Jose, CA, USA; G44-26), PE-conjugated mouse IgG (BD; G155-178), and APC-conjugated mouse IgG (BD; 27–35).
3
Flow Cytometric Analysis of Stem Cell Markers
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Monolayer cells were dissociated with trypsin and blocked in prechilled 2% FBS in PBS for 30 min at 4 °C. Cells were filtered through 70 μm cell strainer and counted. For each sample, 250,000 cells were co-stained with PE-conjugated anti-CD24 and APC-conjugated anti-CD44 or stained with APC-conjugated anti-MUC1 antibodies (BD Bioscience, San Diego, USA) for 30 min on ice. As negative and positive controls, we used non-stained and single-stained cells, respectively. Cells were analysed using Accuri C6 flow cytometer (BD Biosciences) and Flowjo software (Tree Star Inc.).
ALDEFLUORTM Kit (Stemcell Technologies) was used to analyse Aldehyde dehydrogenase (ALDH) enzyme activity, according to the manufacturer’s protocol. A total of 106 cells were incubated with 5 ul ALDH substrate (Bodipy-aminoacetaldehyde) for 45 min at 37 °C. For the negative control, an aliquot of each sample was incubated with diethylaminobenzaldehyde (DEAB). Analysis were performed using Accuri C6 flow cytometer.
ALDEFLUORTM Kit (Stemcell Technologies) was used to analyse Aldehyde dehydrogenase (ALDH) enzyme activity, according to the manufacturer’s protocol. A total of 106 cells were incubated with 5 ul ALDH substrate (Bodipy-aminoacetaldehyde) for 45 min at 37 °C. For the negative control, an aliquot of each sample was incubated with diethylaminobenzaldehyde (DEAB). Analysis were performed using Accuri C6 flow cytometer.
4
Immunoblotting and Flow Cytometry Antibodies
5
Breast Cancer Stem Cell Profiling
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By using a Guava easyCyte flow cytometer (Millipore), the expression of stem cell markers in a breast cancer panel was distinctly evaluated in cells from mammospheres. The antibodies used were phycoerythrin (PE)-conjugated anti-CD24 (BDbiosciences) and fluorescein isothiocyanate (FITC)-conjugated anti-CD44 (BDbiosciences). Staining was done according to the instructions of the manufacturer and analysed in flow cytometer. For the analysis, 80,000 cells were detected and quantified by percentage (%).
6
Stem Cell Characterization Protocol
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Antibodies used were APC-conjugated CD133 antibody (catalog 130-113-184) from Miltenyi Biotec; PE-conjugated anti-CD24 (catalog 555428) from BD Biosciences; FITC-conjugated anti-EpCAM (catalog 60136FI) from StemCell Technologies; anti-SETD1A (catalog A300-289A) from Bethyl Laboratories; anti-PABPC1 (catalog A14872), anti-WDR5 (catalog A3259), anti-CXXC1 (catalog A13423), and anti-ASH2L (catalog A4892) from Abclonal; anti-H3K27ac (catalog ab4729) and anti-H3K27me3 (catalog ab6002) from Abcam; anti–β-actin (catalog AC004) from Abclonal Technology; anti–E-cadherin (catalog 3195S), anti-Vimentin (catalog 5741S), and anti-H3K4me3 (catalog C42D8) from Cell Signaling Technology; and Alexa Fluor Plus 488–conjugated goat anti-rabbit IgG (catalog A32731) and Alexa Fluor 594–conjugated donkey anti-rabbit IgG (catalog A-21207) secondary antibodies from Thermo Fisher Scientific. The detailed information for antibodies is shown in Supplemental Table 3 . DAPI (catalog D9542) was from MilliporeSigma. B27 (catalog A3582801) and N2 supplements (catalog 17502001) were from Gibco. bFGF (catalog 157AA) was from Novoprotein, and EGF (catalog 236-EG) was from R&D Systems.
7
CD133 and CD24 Immunophenotyping
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Cells were dissociated into single cells and counted. Then, 1 × 106 cells were resuspended in 100 μl 2% FBS and stained with APC-conjugated anti-CD133 (Miltenyi Biotec) and PE-conjugated anti-CD24 (BD Biosciences) antibodies on ice for 30 min in the dark. Cells were then washed once and resuspended in staining buffer [0.5 μg/ml DAPI (Sigma) in 2% FBS]. Samples were analyzed on CytoFLEX LX (Beckman) and data analysis was performed using FlowJo software.
8
FACS Isolation and Cell Cycle Analysis of Cancer Stem Cells
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The phycoerythrin (PE)-conjugated anti-CD133 (Miltenyi Biotec), allophycocyanin-conjugated anti-CD44 (BD), PE-conjugated anti-CD24 (BD), or FITC–anti-EpCAM (EMD Millipore) antibodies were used for FACS analysis. To obtain CD133+CD44+ or CD133–CD44– cells, xenografted tumor cells stained with PE-conjugated anti-CD133 and APC-conjugated anti-CD44 antibody (both 1:40) were subjected to cell sorting performed by the FACS. For cell cycle analyses, cells were incubated with 20 µM BrdU (Sigma-Aldrich) for 2 d, prepared as previously described (Tardat et al., 2007 (link)), and incubated for 30 min with PE-conjugated anti-BrdU antibody (R&D Systems).
9
Comprehensive Stemness and EMT Marker Profiling
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COMMD1 (NBP2-4633, Novusbio), ZEB1 (NBP2-13159, Novusbio), Sox2 (ab97959, Abcam), KLF4 (ab151733, Abcam), Oct-4 (2750, Cell Signaling), MYC (5605, Cell Signaling), NANOG (D73G4)(4903, Cell Signaling), K8/18 (20R-Cp004, Fitzgerald Industries), P53 (SC-126, Santa Cruz Biotechnology), Phospho-NFκB p65 (Ser536) (93H1)(3033, Cell Signaling), HIF1A(610959, BD), HIF2A(NB100-122, Novusbio), MCT4(sc-50329, scbt), CAV1(610407, BD), B-actin (A5441, Sigma), tubulin (T4026, Sigma), FITC-conjugated anti-CD24, PE-conjugated anti-CD24, APC-conjugated anti-CD44, APC-conjugated anti-PD-L1 (BD Biosciences).
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