Anti epcam apc

Manufactured by Thermo Fisher Scientific

Anti-EpCAM-APC is a mouse monoclonal antibody conjugated to allophycocyanin (APC). It recognizes the Epithelial Cell Adhesion Molecule (EpCAM) antigen expressed on the surface of epithelial cells.

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6 protocols using anti epcam apc

Multiparametric Liver Cell Sorting

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Human or mouse liver single-cell suspensions were incubated with anti-DLK-FITC (MBL, D187-4, 1:100), anti-EpCAM-APC (eBioscience, 17-5791-82, 1:50), anti-NCAM1 (Sangon Biotech, D198946, 1:50), Alexa Fluor 647 donkey anti-mouse IgG (Invitrogen, A31571, 1:1000), anti-cKit-APC (BioLegend, 105812, 1:200), or anti-CD71-PE (eBioscience, 113808, 1:200) antibodies at 4 °C for 20 min. After washing once with PBS, cells were analyzed and sorted using a FACS Aria SORP cell sorter (BD Biosciences). Dead cells were excluded by DAPI staining.

Wang X., Yang L., Wang Y.C., Xu Z.R., Feng Y., Zhang J., Wang Y, & Xu C.R. (2020). Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level. Cell Research, 30(12), 1109-1126.

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Characterizing OC Stem Cell Markers

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Primary isolated OC were cultured in vitro and passaged for about 6-8 times. The OCs were harvested and then incubated with anti-EpCAM-APC, anti-Sca-1-PE, anti-CD44-PE, anti-CD49f-APC, anti-CD45-APC, anti-CD34-PE, anti-c-kit-FITC, anti-α6 integrin-APC, anti-β1 integrin-PercpCy5.5, anti-β4 integrin-PercpCy5.5 (all from ebioscience, San Diego, CA) at 4°C for 30 mins, after that, cells were fixed with 1% paraformaldehyde and analyzed by a BD Calibur (Becton, Dickinson and Company, New Jersey) and Flowjo software (Tree Star, San Carlos, CA).

Qian L., Zhang H., Gu Y., Li D., He S., Wang H., Cheng Y., Yang W., Yu H., Zhao X., Cai W., Meng L., Jin M., Wang Y, & Zhang Y. (2018). Reduced production of laminin by hepatic stellate cells contributes to impairment in oval cell response to liver injury in aged mice. Aging (Albany NY), 10(12), 3713-3735.

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Lung Cell Isolation and Flow Cytometry

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Single-cell suspensions of lungs isolated from FVB/n;Col1α1-YFP or PyMT;Col1α1-YFP mice were stained using the following antibodies: anti-EpCAM-APC (eBioscience, 17-5791), anti-CD45-PerCP-Cy5.5 (eBioscience, 45-0451), and anti-CD31-PE-Cy7 (eBioscience, 25-0311). DAPI was used to exclude dead cells (Molecular Probes; D3571). Ki67-PE (BioLegend, 652403) intracellular staining of fibroblasts was done using an intracellular staining kit (BD Biosciences, 554714) according to the manufacturer’s protocol. Flow cytometric analysis was performed using CytoFLEX Flow Cytometer (Beckman Coulter). Cell sorting was performed using BD FACSAria II or BD FACSAria Fusion (BD Biosciences). Data analysis was performed with the Kaluza Flow Analysis software (Beckman Coulter).

Shani O., Raz Y., Monteran L., Scharff Y., Levi-Galibov O., Megides O., Shacham H., Cohen N., Silverbush D., Avivi C., Sharan R., Madi A., Scherz-Shouval R., Barshack I., Tsarfaty I, & Erez N. (2021). Evolution of fibroblasts in the lung metastatic microenvironment is driven by stage-specific transcriptional plasticity. eLife, 10, e60745.

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Isolation and Characterization of Mammary Fibroblasts

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Mammary tumours isolated from MMTV-PyMT female mice or normal mammary tissue isolated from 8 weeks old FVB/n female mice were disassociated into single cell suspensions and stained with anti-CD140a-PE (e-Bioscience, 12-1401-81, diluted 1:50) and anti-F4/80-FITC (Cederlane, CL8940F diluted 1:100). DAPI (Molecular probes; D3571, diluted 1:2000) was used to exclude dead cells. For validations, the single cell suspensions were incubated with anti-CD140a-PE, anti-CD45-FITC (eBioscience, 11-0451-81, diluted 1:200), and anti-EpCAM-APC (eBioscience, 17-5791-80, diluted 1:100), and fibroblasts were isolated as CD140a⁺ (PDGFRα⁺) CD45⁻ EpCAM⁻ cells. DAPI was used to exclude dead cells. Sorting was performed using BD FACS Aria II.

Ershaid N., Sharon Y., Doron H., Raz Y., Shani O., Cohen N., Monteran L., Leider-Trejo L., Ben-Shmuel A., Yassin M., Gerlic M., Ben-Baruch A., Pasmanik-Chor M., Apte R, & Erez N. (2019). NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis. Nature Communications, 10, 4375.

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Isolation of Urothelial Subpopulations

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Cells isolated from primary urothelial cells were stained using anti–CD49f-PE antibody (eBiosciences, #12-0495-82) and anti–EPCAM-APC (eBiosciences, #17-5791-82) antibody at a concentration of 5 μL per million cells. The EPCAM⁺/CD49f ^high and EPCAM⁺/CD49f ^low cell population was collected for epithelial transformation assay.

Wang L., Smith B.A., Balanis N.G., Tsai B.L., Nguyen K., Cheng M.W., Obusan M.B., Esedebe F.N., Patel S.J., Zhang H., Clark P.M., Sisk A.E., Said J.W., Huang J., Graeber T.G., Witte O.N., Chin A.I, & Park J.W. (2019). A genetically defined disease model reveals that urothelial cells can initiate divergent bladder cancer phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 117(1), 563-572.

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Flow Cytometry Antibody Panel

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Antibodies for flow cytometry were purchased from eBioscience company as follows: anti-CD45-FITC (11-0451); anti-F4/80-APC (17-4801); anti-MHCII-PE-Cy7 (25-5321); anti-CD11B-PE (12-0112); anti-Ly6G-PercpCy5.5 (45-5931); anti-EpCAM-APC (17-5791); anti-CD24-PE (12-0241); anti-Sca-1-Percp-Cy5.5 (45-5981);anti-CD4-APC (17-0042); anti-Tcrb-PercpCy5.5 (45-5961); anti-IL-17A-PE (12-7178). Cell suspensions were prepared by sieving and gentle pipetting. Cells were washed in ice-cold FACS buffer (2% FCS, 2 mM EDTA in PBS), then incubated with each antibody for 30 min and washed twice with FACS buffer. Data were acquired on a Beckman Gallios flow cytometer and cells were sorted by Beckman Moflo Astrios. Flowjo software was used for data analysis.

Yang D., Chen X., Wang J., Lou Q., Lou Y., Li L., Wang H., Chen J., Wu M., Song X, & Qian Y. (2019). Dysregulated Lung Commensal Bacteria Drive Interleukin-17B Production to Promote Pulmonary Fibrosis through Their Outer Membrane Vesicles. Immunity, 50(3).

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