Anti human epcam

Manufactured by BioLegend

Anti-human EpCAM is a lab equipment product that specifically targets the Epithelial Cell Adhesion Molecule (EpCAM), a transmembrane glycoprotein expressed on the surface of epithelial cells. This product can be used for the identification and isolation of epithelial cells in various applications.

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3 protocols using anti human epcam

Multiparametric Flow Cytometry Profiling

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Cell suspensions were incubated for 20 min at 4 °C in PBS containing 1% FCS and 10 mM EDTA with the following antibodies: anti-mouse CD45, anti-mouse CD31, anti-mouse PDPN, anti-mouse CD21/CD35, anti-mouse SCA1, anti-mouse B220, anti-mouse CD4, anti-mouse CD8, anti-mouse CD19, anti-mouse CD38, anti-mouse GL7 and anti-mouse CD11b (all from BioLegend); anti-mouse CD157 and anti-human CD45 (both from BD Biosciences); anti-human PDPN and anti-human CD31 (both from Thermo Fisher Scientific); and anti-human EPCAM, anti-human CD14, anti-human CD3 and anti-human CD19 (all from BioLegend). LIVE/DEAD cell discrimination was performed either by using a fixable BV510 Dead Cell Staining Kit (Molecular Probes) before antibody staining or by adding 7-aminoactinomycin D (Calbiochem) before acquisition. Cells were acquired with an LSR Fortessa (BD Biosciences) and analyzed with the FlowJo (v.10) software (FlowJo LLC) according to established guidelines. Cell sorting was performed using a BD FACSMelody Cell Sorter and the FACSChorus (v.1.3) software (BD Biosciences).

Lütge M., De Martin A., Gil-Cruz C., Perez-Shibayama C., Stanossek Y., Onder L., Cheng H.W., Kurz L., Cadosch N., Soneson C., Robinson M.D., Stoeckli S.J., Ludewig B, & Pikor N.B. (2023). Conserved stromal–immune cell circuits secure B cell homeostasis and function. Nature Immunology, 24(7), 1149-1160.

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Immune Cell Cytotoxicity Assay

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All target cells were seeded in 96-well flat bottom plates (0.01 x 10 6 cells/well) (#353075, Corning Life Sciences). Effector PBMCs were added to each well at the indicated ratio in PBMC medium. Different concentrations of the respective TCB were added to the wells. The plates were incubated for 72 h.
At the endpoint cells were harvested with Trypsin-EDTA (#25300096, Gibco) and resuspended in FACS buffer (PBS 1x, 2.5 mM EDTA, 1% BSA (#A9647, Sigma-Aldrich), 5% horse serum (#26050, Gibco)) in polypropylene V-bottom 96 well-plates (#651201, Greiner Bio-One). Twenty minutes later, samples were centrifuged and cells were stained with the epithelial cell marker anti-human EpCAM (#324212, BioLegend) at 1:300 concentration in FACS buffer in ice for 30 min. After a wash with 13 PBS, samples were resuspended in the viability marker Zombie Aqua at 1:1000 (#423101, BioLegend) in 1x PBS and acquired on LSR Fortessa (BD Biosciences). Number of alive cells was analyzed with FlowJo software (BD Life Sciences) by means of EpCAM counts.

Martínez-Sabadell A., Morancho B., Rius Ruiz I., Román Alonso M., Ovejero Romero P., Escorihuela M., Chicote I., Palmer H.G., Nonell L., Alemany-Chavarria M., Klein C., Bacac M., Arribas J, & Arenas E.J. (2022). The target antigen determines the mechanism of acquired resistance to T cell-based therapies. Cell reports, 41(3).

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Quantifying Vimentin and EpCAM Expression

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MOS and bulk Matrigel established by Day 7–9 were dissociated into single cells using TrypLE treatment and incubated in 37 °C for 5 minutes. Dissociated cells were washed with PBS + 0.04% BSA and stained with either anti-human Vimentin antibody (CST Cat#, 1:100) combined with PE or anti-human EpCAM (Biolegend Cat#324205, 1:250), at room temperature for 20 minutes. Cells were washed once again with PBS + 0.04% BSA before staining with goat anti-mouse Alexa Fluro 488 secondary antibody (Invitrogen Cat# A32723) for 15 minutes at room temperature. Cells were washed once more with PBS+ 0.04% BSA before flow assay. Sytox blue dead cell stain (A34857) was added as 1:1000 dilution to gate out dead cells in the assays. All flow assays were performed using a Sony SH800 FACS sorter, and flow data were analyzed using FlowJo.

Ding S., Hsu C., Wang Z., Natesh N.R., Millen R., Negrete M., Giroux N., Rivera G.O., Dohlman A., Bose S., Rotstein T., Spiller K., Yeung A., Sun Z., Jiang C., Xi R., Wilkin B., Randon P.M., Williamson I., Nelson D.A., Delubac D., Oh S., Rupprecht G., Isaacs J., Jia J., Chen C., Shen J.P., Kopetz S., McCall S., Smith A., Gjorevski N., Walz A.C., Antonia S., Marrer-Berger E., Clevers H., Hsu D, & Shen X. (2022). Patient-derived micro-organospheres (MOS) enable clinical precision oncology. Cell stem cell, 29(6), 905-917.e6.

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