Cd24 pe

Manufactured by BioLegend

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CD24-PE is a fluorescent-conjugated antibody that binds to the CD24 surface antigen. CD24 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD24-expressing cells using flow cytometry.

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17 protocols using cd24 pe

Isolation and Characterization of Breast Cancer Stem Cells

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Freshly isolated mouse mammary cells were incubated with biotinylated anti-CD31, CD45, and Ter119 cocktail and the labeled Lin⁺ cells were removed by EasySep magnet (StemCell Technologies). The Lin⁻ cells were incubated with fluorescence-conjugated antibodies, including CD24-PE, CD29-APC, and CD49f-FITC antibodies (all from Biolegend), as described (24 (link), 25 (link)). For human cell lines, CD24-PE and CD44-APC antibodies (eBioscience) were used to fractionate the BCSC-enriched population, as described (26 (link)). Isotype antibodies were used as negative controls. Sorting for BCSCs freshly isolated from human breast cancers was performed with Epics Altra flow cytometer (Beckman Coulter). To deplete non-tumor cells from primary cancer samples, a cocktail of lineage marker antibodies including CD2, CD3, CD10, CD16, CD18, CD31, CD64 and CD140b (PharMingen) were used, while to deplete non-tumor cells from mouse specimens, anti-H2 Kd antibody was used.
The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with a high ALDH enzymatic activity, as described (27 (link)). As negative control, for each sample of cells an aliquot was treated with a specific ALDH inhibitor diethylaminobenzaldehyde (DEAB).

Luo M.L., Gong C., Chen C.H., Lee D.Y., Hu H., Huang P., Yao Y., Guo W., Reinhardt F., Wulf G., Lieberman J., Zhou X.Z., Song E, & Lu K.P. (2014). Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer. Cancer research, 74(13), 3603-3616.

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Isolation of Murine Lung Cell Types

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Murine lung tissue was digested into a single-cell suspension as previously described using dispase (Corning catalog #354235), collagenase (Roche catalog #10103578001), and DNase (Roche catalog #10104159001). When isolating AT2 cells from Sftpc^CreERT2:R26R^TdTomato mice, the animals received 200μg/gm tamoxifen (Sigma-Aldrich catalog #T55648) in corn oil (Sigma-Aldrich catalog #C8267) via gavage 3 days prior to being euthanized. Tomato+ cells were isolated using flow cytometry (MoFlow Astrios with Summit v 6.3.0.16900 software). To isolate AT2 cells from mice we stained single-cell suspensions of murine lung tissue and gated based on the following criteria: positive for EpCAM-APC (BioLegend catalog #118213) and negative for CD31-PE (eBioscience catalog #12-0311-81), CD45-PE (eBioscience catalog #12-0451-81), podoplanin-PE (eBioscience catalog #12-5381-80), Sca1-PE (eBioscience catalog #12-5981-81), CD24-PE (BioLegend catalog #119307), and DAPI (BioLegend catalog #422801) as previously described^{81 (link)}. To isolate mesenchymal cells we sorted for cells that were positive for CD140a (BioLegend catalog #135907) and negative for and DAPI (BioLegend catalog #422801). Antibody concentrations are in Supplementary Table 2.

Paris A.J., Hayer K.E., Oved J.H., Avgousti D.C., Toulmin S.A., Zepp J.A., Zacharias W.J., Katzen J.B., Basil M.C., Kremp M.M., Slamowitz A.R., Jayachandran S., Sivakumar A., Dai N., Wang P., Frank D.B., Eisenlohr L.C., Cantu E I.I.I., Beers M.F., Weitzman M.D., Morrisey E.E, & Worthen G.S. (2020). STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury. Nature cell biology, 22(10), 1197-1210.

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Lung Cell Isolation and FACS Analysis

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For FACS analysis, lungs were digested in collagenase (1mg/ml collagenase in 3ml DPBS + 0.2g/L glucose per lung) at 37 °C for 60 min while shaking at 165 rpm, followed by red blood cell lysis with 0.64 % ammonium chloride at 37 °C for 3 min [31 (link)]. Cells were resuspended in blocking solution (anti-FcR and Rat IgG) and incubated on ice for 10 min. Cells were stained with conjugated antibodies CD31-PECy7, CD45-PECy7, EpCAM-APC, CD104-FITC and CD24-PE (Biolegend) as described previously [32 (link)]. Cells were then washed and resuspended in PI solution. Cells were sorted on an ARIA II (Beckton Dickinson).

Holik A.Z., Filby C.E., Pasquet J., Viitaniemi K., Ciciulla J., Sutherland K.D, & Asselin-Labat M.L. (2015). The LIM-domain only protein 4 contributes to lung epithelial cell proliferation but is not essential for tumor progression. Respiratory Research, 16(1), 67.

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Immunophenotypic Characterization of Cells

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SCAP aliquots (1 × 10⁶ cells) were used to detect the immunophenotype according to a previously developed protocol [35 (link)]. The following anti-human monoclonal antibodies were used: CD45/FITC (Cat#304005, BioLegend, San Diego, CA, USA), CD34/FITC (Cat# 343603, BioLegend), CD90/FITC (Cat#328107, BioLegend), CD146/FITC (Cat#361011, BioLegend), CD44/FITC (Cat#338803, BioLegend), and CD24/PE (Cat#311105, BioLegend). Non-specific IgG/FITC (Cat#400107, BioLegend) or IgG/PE (Cat#400211, BioLegend) was used to be the isotype control. The data were analyzed using a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Liu Z., Lin Y., Fang X., Yang J, & Chen Z. (2021). Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP–Smad Signaling Pathway. Molecules, 26(6), 1580.

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Comprehensive B Cell Immunophenotyping

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Flow cytometric analysis was performed on BD® LSR II Flow Cytometer (Marshallscientific, USA), and data were analyzed with FlowJo10.0 software (Treestar, Ashland, OR, USA). Anti- mouse B220-FITC (103205, Biolegend, USA), CD43-PE-Cy7 (143210, Biolegend, USA), CD24-PE (101807, Biolegend, USA), CD19-PE-Cy7 (552854, BD, USA), CD19-BV421 (115520, Biolegend, USA), CD19-PE (115508, Biolegend, USA), CD69-FITC (104506, Biolegend, USA), CD5-PE (100608, Biolegend, USA), CD86-APC (105011, Biolegend, USA), CD95-PE (152608, Biolegend, USA), BP-1-Alexa Fluor 647 (108312, Biolegend, USA), IgD-APC (405714, Biolegend, USA), IgM-PE (406507, Biolegend, USA), and corresponding isotype control antibodies were purchased from Biolegend. Antibodies were listed in Supplementary Table 3.

Kang X., Chen S., Pan L., Liang X., Lu D., Chen H., Li Y., Liu C., Ge M., Zhang Q., Liu Q, & Xu Y. (2022). Deletion of Mettl3 at the Pro-B Stage Marginally Affects B Cell Development and Profibrogenic Activity of B Cells in Liver Fibrosis. Journal of Immunology Research, 2022, 8118577.

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Tumor Dissociation and Flow Cytometry

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Tumors were collected in cold DMEM supplemented with 2.5% FCS and finely chopped using a surgical scalpel before enzymatic dissociation using collagenase type 4 (Worthington Biochemical Corp, Lakewood, NJ, USA) to obtain single cell suspensions for flow cytometry. Cells were then stained with BD Horizon fixable viability stain 780, CD44 APC, and CD24 PE (BioLegend, San Diego, CA) and acquired using a BD LSR II instrument. Results were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Dunn J., McCuaig R.D., Tan A.H., Tu W.J., Wu F., Wagstaff K.M., Zafar A., Ali S., Diwakar H., Dahlstrom J.E., Bean E.G., Forwood J.K., Tsimbalyuk S., Cross E.M., Hardy K., Bain A.L., Ahern E., Dolcetti R., Mazzieri R., Yip D., Eastgate M., Malik L., Milburn P., Jans D.A, & Rao S. (2022). Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers. Cancers, 14(6), 1596.

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CD24 and CD44 Expression in Plated Cells

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Plated cells at about 60% confluence were grown on Lab-Tek 2-chamber glasses (Nunc) in the normal culture medium under standard culture conditions. The cells were fixed with 3.7% formaldehyde solution for 15 min, followed by washing with PBS three times and incubation in PBS with 0.1% BSA for 45 min. After washing with PBS, the cells were incubated for 45 min with anti-human antibodies CD24 PE (BioLegend, 311106) and CD44 APC (BioLegend, 338806) diluted (1:20) in PBS with 0.1% BSA. The CD24 and CD44 specimens were mounted on microscope slides, and expression was observed with an LSM 880 AiryScan (Zeiss).
For the exosome treatment experiment, s-TME CMFDA/DMSO-Exo and s-TME CMFDA/GW4869-Exo were treated onto Panc0203 cells and maintained in the standard culture condition for 20 h.

Jang G., Oh J., Jun E., Lee J., Kwon J.Y., Kim J., Lee S.H., Kim S.C., Cho S.Y, & Lee C. (2022). Direct cell-to-cell transfer in stressed tumor microenvironment aggravates tumorigenic or metastatic potential in pancreatic cancer. NPJ Genomic Medicine, 7, 63.

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Isolation and Characterization of Adipose Progenitor Cells

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Isolated stromal-vascular fraction was resuspended in FACS media (2% FCS/PBS/0.01% NaN₃) and cells were stained with markers for adipose stem cell and preadipocytes (Rodeheffer et al., 2008 (link)). Antibody incubation was performed on ice for 20 min and fixed with 1% paraformaldehyde overnight at 4°C. Samples were acquired on Canto II flow cytometry (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc., Ashland, OR). For all ex vivo analysis, PDZ-RhoGEF expression, progenitor cell proliferation, and PI3K signaling, adipogenic progenitor cells were sorted from adipogenic progenitor specific maker-labeled SVF suspensions with AriaII (Becton Dickson) cell sorter, and the purity of sorted cells was verified as >95% by rerunning the sorted population. Antibodies were purchased from eBioscience unless otherwise stated, including the following: biotin-lineage cocktail (), Sca1-Pacific Blue (Biolegend, San Diego, CA), Sca1-APC, CD34-Alexa-Fluro-700 (Biolegend), CD24-PE, CD31-PE-Cy7, Terr-119-APC-eFluro-780, biotin-CD45/streptavidin-PerCp, CD29-FITC (BD Biosciences), streptavidin-PE-Cy7, and anti-Dlk/Pref-1 (MBL).

Chang Y.J., Pownall S., Jensen T.E., Mouaaz S., Foltz W., Zhou L., Liadis N., Woo M., Hao Z., Dutt P., Bilan P.J., Klip A., Mak T, & Stambolic V. (2015). The Rho-guanine nucleotide exchange factor PDZ-RhoGEF governs susceptibility to diet-induced obesity and type 2 diabetes. eLife, 4, e06011.

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Flow Cytometry Analysis of Stem Cell Markers

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Cells were trypsinized (0.05% Trypsin), counted, washed twice with 1xPBS, and resuspended in 50ul 1%BSA in PBS. Cells were incubated with 5ul (anti-CD44) and 2ul (anti-CD24) antibody for 45 min on ice. Then washed with 1%BSA in PBS twice and resuspended in 1ml 1xPBS. Tubes were then read on Guava easyCyte flow cytometer. All data were collected and analyzed using a Guava EasyCyte Plus system and CytoSoft (version 5.3) software (Millipore). Data are gated and expressed relative to the appropriate unstained and single stained controls. Antibodies were purchased from; CD44-FITC (BD Biosciences; 555478), CD24-PE (Biolegend; 31106), CD24-PECy5 (Biolegend; 101812). For NANOG-GFP experiments, cells were stably transduced with NANOG-GFP (Addgene) and analyzed on GFP channel after respective treatments. The Aldeflour assay was performed using the ALDEFLOUR kit from STEMCELL Technologies as per manufacturer’s instructions. Unstained and single-stained controls were used for gating.

Akella N.M., Le Minh G., Ciraku L., Mukherjee A., Bacigalupa Z.A., Mukhopadhyay D., Sodi V.L, & Reginato M.J. (2020). O-GlcNAc Transferase Regulates Cancer Stem-like Potential of Breast Cancer Cells. Molecular cancer research : MCR, 18(4), 585-598.

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Mammary Gland Cell Isolation and FACS

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Mammary glands of CO (vehicle)-treated and 4OHT-treated 8- to 9-week-old virgin littermate female FVB/Tfap2c^fl/fl/Krt5-Cre-ER^T2 mice were digested in a mixture of gentle collagenase/hyaluronidase (STEMCELL, cat. no. 07919) with 5% fetal bovine serum (FBS) in complete EpiCult-B (STEMCELL, cat. no. 05611) media for 15 h at 37°C. The red blood cells were lysed in NH₄Cl. The cell suspension was further digested in 0.25% trypsin-EDTA for 1 min then 5 U/mL dispase (STEMCELL, cat. no. 07913) plus 0.1 mg/mL DNase I (STEMCELL, cat. no. 07900) for 1 min followed by filtration through a 40-μm cell strainer. After washing in Hank’s balanced salt solution, cell viability was assessed by trypan blue. Single-cell isolation procedure was performed as described previously (Borcherding et al., 2015 (link)). FACS was carried out using FACS Aria (Becton Dickinson). The Lin^– population was defined as TER119^–, CD31⁻, and CD45^–. FACS data were analyzed using FlowJo software (v.10.1r7, Tree Star). The following antibodies were used: CD31-Biotin (BioLegend, 102404, 1:250), TER-119-Biotin (BioLegend, 116204, 1:250), CD45-Biotin (BioLegend, 103014, 1:250), Strep-PE-Cy7 (Invitrogen, 25-4317-82, 1:250), CD24-PE (BioLegend, 101807, 1:250), CD49f-FITC (BioLegend, 313606, 1:200).

Gu V.W., Cho E., Thompson D.T., Cassady V.C., Borcherding N., Koch K.E., Wu V.T., Lorenzen A.W., van der Heide D.M., White J.R., Kulak M.V., Williams T., Zhang W, & Weigel R.J. (2020). AP-2γ Is Required for Maintenance of Multipotent Mammary Stem Cells. Stem Cell Reports, 16(1), 106-119.

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