Human peripheral blood mononuclear cells from the G-CSF-treated healthy volunteer were stained in an AutoMACS Running Buffer in 4°C with the following antibodies: cocktail of antibodies for hematopoietic lineage markers (Lin) (anti-CD2-FITC (#555326, clone RPA2.10), anti-CD3-FITC (#555332, clone UCHT1), anti-CD14-FITC (#345784, clone M5E2), anti-CD16-FITC (#555406, clone 3G8), anti-CD19-FITC (#555412, clone HIB19), anti-CD24-FITC (#555427, clone ML5), anti-CD56-FITC (#345811, clone NCAM16.2), anti-CD66b-FITC (#555724, clone G10F5), and anti-CD235a-FITC (#559943, clone GA-R2(HIR2), all 1 : 200) all from BD Pharmingen), anti-CD45-APC-Cy7 (#304014, clone 2D1, BD Biosciences, 1 : 50), anti-CD34-APC or anti-CD34-FITC (#555824, #555821, clone 581, BD Pharmingen, 1 : 50), anti-CD133-PE (#130-080-801, clone AC133, Miltenyi Biotec, 1 : 50), anti-CD105-PE (#323206, clone 43A3, BioLegend, 1 : 50), anti-CD90-APC (#559869, clone 5E10, BioLegend), anti-CD181-APC (#551080, clone 5A12, BD Pharmingen), anti-CD184(CXCR4)-APC (#555976, clone 12G5, BioLegend, 1 : 50), anti-CD11b-APC (#101211, clone M1/70, BioLegend, 1 : 50), and 10 μg/mL Hoechst 33342 (B2261-25MG, Sigma Aldrich). Stained cells were then analyzed on a BD LSR II flow cytometer using BD FACS Diva software (Becton Dickinson). Controls for gating are shown in Supplementary Figure 1 .
Anti cd24 fitc
Manufactured by BD
Sourced in United States
Anti-CD24-FITC is a fluorescent-labeled antibody that binds to the CD24 cell surface antigen. It is used for the identification and analysis of CD24-expressing cells in flow cytometry applications.
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Lab products found in correlation
Anti cd44 pe, by BD (8 mentions)
Facsaria, by BD (3 mentions)
Anti cd14 fitc, by BD (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Anti cd45 pe, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti il 10 pe, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Anti cd19 fitc, by BD (1 mentions)
Anti cd56 fitc, by BD (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Anti cd66b fitc, by BD (1 mentions)
Anti cd11b apc, by BioLegend (1 mentions)
Anti cd45 apc cy7, by BD (1 mentions)
Anti cd2 fitc, by BD (1 mentions)
Anti cd105 pe, by BioLegend (1 mentions)
Anti cd90 apc, by BioLegend (1 mentions)
Anti cd16 fitc, by BD (1 mentions)
Anti cd133 pe, by Miltenyi Biotec (1 mentions)
18 protocols using anti cd24 fitc
1
Multiparametric Analysis of Human Hematopoietic Cells
2
Identifying Cancer Stem Cell Phenotype in GCa Cells
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To identify the cancer stem cell phenotype of GCa cells, anti-CD24-FITC, anti-CD24-PE, anti-CD24-APC, anti-CD44-PE, anti-CD45-PE (BD Pharmingen, San Diego, CA, USA), anti-CD133/2-APC (MACS Miltenyi Biotec, Teterow, Germany) were used. Dead cells were detected with 7-AAD (BD Pharmingen). All flow cytometry data were acquired on BD FACS Aria for cell sorting, FACS Canto and FACS Calibur (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Tree Star, San Carlos, CA, USA).
3
Quantification of CD19+ B Cells
4
Isolation and Characterization of Cancer Stem Cells
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Cells were trypsinized into single cell suspension and were counted. For staining, samples were usually incubated with antibodies for 30 minutes at 4°C. Unbound antibody was washed off and cells were sorted by flow cytometry no longer than 30 min post staining on a BD Aria III. In order to isolate breast CSCs, the antibodies used were anti-CD44 PE and anti-CD24 FITC (BD Pharmingen). The purity of isolated breast CSCs was determined by standard flow cytometry analysis. The purity of isolated CD44+CD24– CSCs regularly exceeded 98%. As for sorting pancreatic CSCs, cells were stained with APC-labeled CD44 antibodies and PE-labeled CD24 antibodies (BD Pharmingen), as well as BV421-labeled ESA antibodies (Biolegend). The triple positive (CD44+CD24+ESA+) cells and L3.6pl were reanalyzed by flow cytometry analysis using PE-labeled Sox2 antibodies (Biolegend). In sorted cell implantation experiments, CSCs were isolated using APC-labeled antibody against human CD133 (Miltenyi Biotech).
5
Multiparametric Flow Cytometry of Immune Cells
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Flow cytometry was performed with the following antibodies: anti-CD19-Percp-Cy5.5, anti-CD19-Percp-eFluor710, anti-CD24-FITC, anti-CD38-superbright600, anti-CD38-PE, anti-Ki-67-Alexa Fluor647, anti-IL-10-PE, anti-TIM-1-PE, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-17A-APC (eBioscience, San Diego CA, USA), anti-IL-6-APC, anti-TNF-α-FITC, anti-IL-12-BV-421, and anti-CD24-Alexa Fluor647 (BD Biosciences, France). Intracellular cytokines and Ki-67 were assessed in cells treated with Permeabilization and IC Fixation Buffers (eBioscience, San Diego, CA, USA). Stained cells were analyzed with a 21-color ZE5 cell analyzer (Bio-Rad, USA). The CD19+CD24hiCD38hi B subset was sorted (FACSAria; BD Pharmingen) using anti-CD19-Percp-Cy5.5, anti-CD24-FITC, and anti-CD38-PE. The sort purity of CD19+CD24hiCD38hi B cells was routinely >90%. CD4+T subpopulation was sorted (FACSAria; BD Pharmingen) using anti-CD38-PE and anti-CD3-PE. The sort purity of CD4+T cells was routinely >95%. All flow cytometry data were analyzed with FlowjoV10 Software (Tree Star, OR, USA).
6
Characterization of Mammosphere and Tumor Cells
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Mammospheres were trypsinized into single-cell suspension with Accutase-Enzyme Cell Detachment Medium, and then washed and resuspended in PBS. Single cells were labeled with antibodies specific for human cells: anti-CD24-FITC and anti-CD44-PE (BD PharmingenTM). An unstained, single stain served as the control. Isotype controls were used to exclude non-specific conjunctions. After being incubated with antibodies for 30 min at 4°C in the dark, the unbound antibody was washed. Cells were fixed for analysis (FC500MCL, Beckman Coulter, USA).
For tumor analysis, harvested tumors were minced to form single cell suspension. The tissue lysate was filtered through a 200 mesh sieve prior to stain. Surface antigen CD24 and CD44 were detected as above. The single cell suspension was also observed under a light microscope.
For tumor analysis, harvested tumors were minced to form single cell suspension. The tissue lysate was filtered through a 200 mesh sieve prior to stain. Surface antigen CD24 and CD44 were detected as above. The single cell suspension was also observed under a light microscope.
7
Isolation and Analysis of Lung Tumor Cells
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Mice were euthanized with avertin overdose and lungs were dissected and examined grossly for tumor formation. Tumors were dissected from the lungs of primary mice and tumor tissue was prepared as described2 (link). Briefly, tumors were isolated, minced, digested rotating for 1 h at 37 °C with 2 mg/ml collagenase/dispase (Roche), and then filtered twice (100 μm, and then 40 μm) after a 5 min incubation with 0.025 mg/ml DNase. Cell lines were trypsinized and then filtered (40 μm). Single-cell suspensions were stained using anti-CD31-APC (BD Pharmingen 551262), anti-CD45-APC (BD Pharmingen 559864), anti-Epcam-PE-Cy7 (BioLegend 118216), anti-CD24-FITC (BD Pharmingen 553261), anti-Sca-1-APC-Cy7 (BD Pharmingen 560654), with DAPI (Sigma) staining to visualize dead cells. All antibodies were incubated for 15–20 min at 1:100 dilutions. Cell sorting was performed with a Beckman Coulter/Cytomation MoFlo or a BD FACS Aria, and data were analyzed with the FlowJo software (Tree Star Inc.). Example gating strategy is illustrated in Supplementary Figure 9 .
8
Quantifying Cancer Stem Cells via ALDH1 and CD44/CD24
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Aldehyde dehydrogenase 1 (ALDH1) enzyme activity was assayed with the Aldefluor assay kit (StemCell Technologies) following the manufacturer's instructions. TGFβ1-stimulated and transfected cells were incubated with the Aldefluor reagent for 45 min at 37 °C or DEAB (diethylaminobenzaldehyde) as the negative control. CD44⁺/CD24-/low cell populations were also evaluated by staining with anti-CD44-PE and anti-CD24-FITC (BD Pharmingen) or their isotype controls at 4 °C for 15 min. The effect of LY and/or CHIR was assessed in 2MS after treatment with the inhibitors and TGFβ1 for 72h. ALDH1+ and CD44⁺/CD24-/low subpopulations were analyzed in a FACSVerse (BD Biosciences) flow cytometer. The side population was determined by Hoechst 33342 dye exclusion assay. Briefly, after stimulation with TGFβ1 and transfection with siRNA, cells were incubated for 90 min at 37 °C in DMEM supplemented with 2% FBS, 10 mM HEPES, and 5 µg/ml Hoechst 33342 (Sigma-Aldrich) in the dark with interval mixing. Verapamil (50 µM) was used as a control of inhibition. Hoechst 33342 was excited with a UV laser at 355 nm, and emissions were detected at 450/50 nm (Hoechst blue) and 670/30 nm (Hoechst red) in a FACSAria III cell sorter (BD Biosciences).
9
Characterization of Stem Cell Markers
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Cells at 70%–85% confluence were dissociated with trypsin (0.25%,Sigma-Aldrich), incubated in PBS, then blocked with 3% bovine serum albumin (BSA) for 30 minutes. Cells (1 × 106) were stained with antibodies against CD24 (anti CD24-FITC; BD Pharmingen, Franklin Lakes, NJ, USA), CD133 (anti CD133-FITC; Miltenyi Biotec, Germany), CD47 (anti CD47-FITC; Santa Cruz Biotechnology, USA), CD29 (anti CD29-PE; BD Pharmingen), CD44 (anti CD44-PE; BD Pharmingen), CXCR4 (anti CXCR4-PE; Beckman Coulter, USA), SSEA3 (anti SSEA3-FITC; BD Pharmingen), and SSEA4 (anti SSEA4-PE; BD Pharmingen), then washed twice with PBS and analyzed using the FACS Canto II system (BD Biosciences, USA).
10
Dissecting IL-10 Producing B Cells
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After 4 h of stimulation, PBMCs were stained with a combination of the following antibodies from BD Bioscience: anti-CD19-PerCP (cat #345–778), anti-CD43-FITC (cat #555–475), anti-CD27-PECy7 (cat #560–609), anti-CD24-FITC (cat #555–427), anti-CD38-PECy7 (cat #335–825), anti-CD25 FITC (cat #555–431), anti-IL-10 APC (cat #554–707) or anti-IL-10-PE (cat #559–330). Anti-CD5 APC was from Dako (cat #C7242, Glostrup, Denmark) and anti-TIM-1 PE was purchased from Biolegend (cat #353–904).
After 48 h of stimulation, IL-10 was detected with the following BD Bioscience Abs: anti-CD19 APC (cat #555–415), anti-CD14 FITC (cat #555–397), anti-CD4 PerCP (cat #345–770), anti-CD8 PECy7 (cat #557–746) and anti-IL-10 PE (cat #559–330). Live/Dead Fixable Near InfraRed staining (cat #L10119; Molecular Probes, Invitrogen, Carlsbad, CA) was included.
The cells were acquired with a FACS Canto (BD Bioscience) flow cytometer with argon laser (488 nm) and Helium-Neon laser (633nm) excitation.
All analyses were carried out using FlowJo V10 (TreeStar, Ashland, OR). Dead cells were excluded based on Live/Dead Fixable Near InfraRed staining and B cells were identified as CD19+ events within a morphological lymphocyte gate. Individual IL-10+ B cells were identified using the gating strategy demonstrated inS1 Fig .
After 48 h of stimulation, IL-10 was detected with the following BD Bioscience Abs: anti-CD19 APC (cat #555–415), anti-CD14 FITC (cat #555–397), anti-CD4 PerCP (cat #345–770), anti-CD8 PECy7 (cat #557–746) and anti-IL-10 PE (cat #559–330). Live/Dead Fixable Near InfraRed staining (cat #L10119; Molecular Probes, Invitrogen, Carlsbad, CA) was included.
The cells were acquired with a FACS Canto (BD Bioscience) flow cytometer with argon laser (488 nm) and Helium-Neon laser (633nm) excitation.
All analyses were carried out using FlowJo V10 (TreeStar, Ashland, OR). Dead cells were excluded based on Live/Dead Fixable Near InfraRed staining and B cells were identified as CD19+ events within a morphological lymphocyte gate. Individual IL-10+ B cells were identified using the gating strategy demonstrated in
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