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Epcam g8

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EpCAM (G8.8) is a mouse monoclonal antibody that recognizes the human epithelial cell adhesion molecule (EpCAM) antigen. EpCAM is a transmembrane glycoprotein involved in cell-cell adhesion in epithelial tissues.

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Lab products found in correlation

Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions) Cd45 30 f11, by BioLegend (3 mentions) Cd8β yts156, by BioLegend (3 mentions) Fixation and permeabilization buffer, by BioLegend (2 mentions) Fc block, by Thermo Fisher Scientific (2 mentions) Il 22 1h8pwsr, by Thermo Fisher Scientific (2 mentions) Il 13 ebio13a, by Thermo Fisher Scientific (2 mentions) Cell stimulation cocktail, by Thermo Fisher Scientific (2 mentions) Fixable live dead stain, by Thermo Fisher Scientific (2 mentions) Dithiothreitol (dtt), by Merck Group (2 mentions) Dnase, by Merck Group (2 mentions) Dispase, by Merck Group (2 mentions) Facsaria 2, by BD (2 mentions) Ab96926, by Abcam (2 mentions) Cd103 m290, by BD (2 mentions) Cd11c hl3, by BD (2 mentions) B220 ra3 6b2, by Thermo Fisher Scientific (2 mentions) Avidin biotin blocking reagent, by Vector Laboratories (2 mentions) Cd90 1 his5, by Thermo Fisher Scientific (2 mentions) Ab26120, by Abcam (2 mentions)

Close spelling variants of this product

EpCAM (75 citations) EpCAM clone G8.8 (8 citations) Anti-EpCAM (G8.8; (6 citations) Allophycocyanin-conjugated anti-EpCam (clone G8.8; (4 citations) Anti-EpCAM (clone G8.8; (3 citations) PE/Cyanine7 anti-mouse CD326 (EpCAM) antibody (G8.8, (2 citations) EPCAM PE/Cy7 clone G8.8 (2 citations) Anti-EpCam-AF647/BUV421 (G8.8; (2 citations) EpCAM(G8.8)-PeCy7 (2 citations)

12 protocols using epcam g8

1

Isolation and Enrichment of Tumor Stromal Cells

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Tumor organoids were isolated from primary tumors as described previously (5 (link)), with the ECM prepared as in (16 (link)). Stromal cells were isolated during the differential centrifugations that are used to separate organoids from the single cells. The supernatant from these centrifugation steps was collected and centrifuged at 1500 rpm for 5 minutes. The cell pellet was re-suspended in cell depletion buffer (PBS + 2% FCS + 1 mM EDTA), counted and diluted to 1×108 cells/ml. Biotin conjugated antibodies targeting CD326 (EpCAM; G8.8; 2 μg/ml; Biolegend), CD45 (30-F11; 2 μg/ml; Biolegend) and erythroid cells (TER-119; 1 μg/ml; STEMCELL Technologies) were used to deplete epithelial/immune/erythroid cells using the EasySep™ Mouse Streptavidin RapidSpheres™ Isolation Kit (STEMCELL Technologies), per the manufacturer’s instructions. The efficiency of cell depletion was assessed by flow cytometry using standard protocols. Details of this analysis are provided in Supplementary Materials and Methods.
Hanley C.J., Henriet E., Sirka O.K., Thomas G.J, & Ewald A.J. (2020). Tumor resident stromal cells promote breast cancer invasion through regulation of the basal phenotype. Molecular cancer research : MCR, 18(11), 1615-1622.
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2

Isolation and Characterization of Colonic Immune Cells

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Lamina propria cells from colonic tissue were harvested as before8 (link). For intracellular cytokine staining, cells were stimulated using the eBioscience cell stimulation cocktail for 4 h at 37 °C. Cells were fixed and permeabilized using the Biolegend fixation and permeabilization buffer. The following antibodies (clones) were used for staining: CD45 (30-F11), CD19 (6D5), CD11b (M1/70), CD90.2 (53–2.1), CD3 (145–2C11), TCR-β (H57–597), CD4 (RM4–5), CD8 (53–6.7), Gr-1 (RB6–8C5), MHCII (M5/114.15.2), CD11c (N418), CD103 (2E7), IFN-γ (XMG1.2) from Biolegend and IL-22 (1H8PWSR), and IL-13 (eBio13A) from eBioscience. All samples were blocked with Fc block (TruStainfcx) and stained with a fixable live dead stain (Invitrogen). FlowJo v.10 was used to analyze flow cytometry data. Intestinal epithelial cells (IECs) were harvested by incubation at 37°C with 2mM DTT (Sigma) followed by two incubations with 5mM EDTA and then digested with Dispase (Sigma) and DNase (Sigma). IECs were sorted as PI−CD45−EpCam+ using the following antibody clones: CD45 (30-F11) and EpCam (G8.8) from Biolegend on an FACSAria II (BD Biosciences). Purity of IECs was ≥ 95%.
Neil J.A., Matsuzawa-Ishimoto Y., Kernbauer-Hölzl E., Schuster S.L., Sota S., Venzon M., Dallari S., Galvao Neto A., Hine A., Hudesman D., Loke P., Nice T.J, & Cadwell K. (2019). IFN-I and IL-22 mediate protective effects of intestinal viral infection. Nature microbiology, 4(10), 1737-1749.
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3

Multiparameter Flow Cytometry Antibodies

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Fluorochrome conjugated antibodies against human CD45 (clone H130), CD49a (SR84), CD16 (3G8), and CD14 (M5E2) were purchased from BD Biosciences. Antibodies against human CD3 (OKT3), PD-1 (EH12.2H7), Granzyme A (CB9), CD56 (HCD56), CD49a (TS2/T), and CD103 (Ber-ACT8) were purchased from Biolegend. Anti-human CD15 (MMA) was purchased from eBioscience, now Thermo Scientific, and anti-human NKG2A (REA110) was purchased from Miltenyi Biotec. Anti-human CD8α (RPA-T8) and biotinylated anti-human CD3 (UCHT1) were purchased from Tonbo Biosciences. Fluorochrome conjugated antibodies against mouse CD45 (clone 30-F11), CD49a (Ha31/8), CD103 (M290), Ly6G (1A8), F4/80 (T45–2342), CD11b (M1/70), MHC class II A-A/I-E (M5/114/15/2), and CD11c (N418) were purchased from BD Biosciences. Fluorochrome conjugated antibodies against mouse CD3ε (17A2), NK1.1 (PK136), CD19 (D1/CD19), XCR1 (ZET), CD49b (DX5), Ter119 (Ter-119), CD29 (HmB1–1), and EpCAM (G8.8) were purchased from BioLegend. Fluorochrome conjugated antibodies against mouse/human granzyme B (GB11) was purchased from Invitrogen. Fluorochrome conjugated antibodies against mouse CD27 (LG.7F9), CD31 (390), and CD24 (M1/69) were purchased from eBioscience.
Kansler E.R., Dadi S., Krishna C., Nixon B.G., Stamatiades E.G., Liu M., Kuo F., Zhang J., Zhang X., Capistrano K., Blum K.A., Weiss K., Kedl R.M., Cui G., Ikuta K., Chan T.A., Leslie C.S., Hakimi A.A, & Li M.O. (2022). Cytotoxic Innate Lymphoid Cells Sense Cancer Cell-Expressed Interleukin-15 to Suppress Human and Murine Malignancies. Nature immunology, 23(6), 904-915.
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4

Quantitative Immunofluorescence Imaging of Intestinal Tissues

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Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7-8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking reagent when necessary (Vector labs) and stained with the following biotinylated or directly conjugated antibodies: CD8β (YTS156.7.7, Biolegend), CD4 (RM4-5, eBioscience), CD103 (M290, BD Biosciences), CD90.1 (HIS5.1, eBioscience), Epcam (G8.8, Biolegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, eBioscience). Rabbit anti-Yersinia pseudotuberculosis (ab26120, Abcam) and anti-rabbit Dylight 649 (ab96926, Abcam) were used to stain for bacterial antigens. Stained slides were mounted with Prolong Gold antifade reagent (Invitrogen), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.
The number of OT-I cells/villus was determined by sectioning a ‘Swiss roll’52 (link) of the distal third of the small intesine. Five or more sections/mouse that were at least 400μm apart were stained and imaged. A villus and the underlying submucosa and muscularis were considered a single villus, and the number of OT-I cells in each region was determined. The number of OT-I cells/villus were binned and plotted as the percentage of villi containing a given range of OT-I cells.
Bergsbaken T, & Bevan M.J. (2015). Proinflammatory microenvironments within the intestine regulate differentiation of tissue-resident CD8 T cells responding to infection. Nature immunology, 16(4), 406-414.
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5

Multicolor Flow Cytometric Analysis

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For detection and analysis, the following antibodies/clones were used: FcɛR1a (MAR-1; BioLegend); CD117 (2B8; BioLegend); CD45 (30-F11; BioLegend), β7 integrin (M293; BD Biosciences); CD11b (M1/70; BioLegend); CD11c (N418; BioLegend); CD4 (GK1.5; BioLegend); CD8 (53–6.7; BioLegend); CD103 (2E7; BioLegend); EPCAM (G8.8; BioLegend); Thy1.2 (53–2.1; BioLegend); CD31 (390; eBioscience); TGF-β1 (Tw7-16B4; BioLegend); and mMCP-1 (RF6.1; eBioscience). Anti-mMCP-1 and isotype control were conjugated in parallel to Alexa Fluor 647 using a conjugation kit (Life Technologies). Intracellular staining was conducted using a BD Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer-supplied protocol. For flow cytometry, the cells were collected and stained for surface markers for 45 min before fixation. Intracellular mMCP-1 staining was conducted overnight at 4°C. All cell sorting was on a BD FACSAria Fusion cell sorter using BD FACSDiva software. For all flow cytometry not involving cell sorting, data were collected on a BD LSRII Fortessa or BD CANTO-II using BD FACSDiva software. All downstream data analysis was conducted in FlowJo.
Derakhshan T., Samuchiwal S.K., Hallen N., Bankova L.G., Boyce J.A., Barrett N.A., Austen K.F, & Dwyer D.F. (2020). Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation. The Journal of Experimental Medicine, 218(1), e20200321.
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6

Multiparametric Analysis of Intestinal Immune Cells

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Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7–8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking (Vector labs) and stained with the following biotinylated or directly conjugated antibodies:
CD8β (YTS156.7.7, Biolegend), CD45.2 (104, eBioscience), Epcam (G8.8, Biolegend), CD11b (M1/70, eBioscience), and CD11c (HL3, eBioscience). For identification of YFP-producing cells, sections were stained with anti-GFP (Abcam, ab6556), anti-rabbit-FITC (Abcam, ab6108), and anti-FITC-AlexaFlour488 (Invitrogen, polyclonal) antibodies. No reactivity was observed in infected YFP-negative mice. Stained slides were mounted with Prolong Gold antifade reagent (Thermo Fisher Scientific), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.
Bergsbaken T., Bevan M.J, & Fink P.J. (2017). Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells. Cell reports, 19(1), 114-124.
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7

Multicolor Flow Cytometry of Immune Cells

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Membrane and intracellular staining of MLN or epithelial cells were performed as described.22 The following antibodies were used: CD4 (GK1.5, BioLegend), CD8α (53–6.7, BD Biosciences), CD16/CD32 (93, BioLegend), IL-17A (TC11-18H10.1, BioLegend), RORγt (Q31-378, BD Biosciences), IFNγ (XMG1.2, BD Biosciences), IL-10RA (1B1.3a, BioLegend), and Ep-CAM (G8.8, BioLegend).
Bhutiani N., Li Q., Anderson C.D., Gallagher H.C., De Jesus M., Singh R., Jala V.R., Fraig M., Gu T, & Egilmez N.K. (2018). Enhanced gut barrier integrity sensitizes colon cancer to immune therapy. Oncoimmunology, 7(11), e1498438.
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8

Quantitative Immunofluorescence Imaging of Intestinal Tissues

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Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7-8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking reagent when necessary (Vector labs) and stained with the following biotinylated or directly conjugated antibodies: CD8β (YTS156.7.7, Biolegend), CD4 (RM4-5, eBioscience), CD103 (M290, BD Biosciences), CD90.1 (HIS5.1, eBioscience), Epcam (G8.8, Biolegend), CD11c (HL3, BD Biosciences), B220 (RA3-6B2, eBioscience). Rabbit anti-Yersinia pseudotuberculosis (ab26120, Abcam) and anti-rabbit Dylight 649 (ab96926, Abcam) were used to stain for bacterial antigens. Stained slides were mounted with Prolong Gold antifade reagent (Invitrogen), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.
The number of OT-I cells/villus was determined by sectioning a ‘Swiss roll’52 (link) of the distal third of the small intesine. Five or more sections/mouse that were at least 400μm apart were stained and imaged. A villus and the underlying submucosa and muscularis were considered a single villus, and the number of OT-I cells in each region was determined. The number of OT-I cells/villus were binned and plotted as the percentage of villi containing a given range of OT-I cells.
Bergsbaken T, & Bevan M.J. (2015). Proinflammatory microenvironments within the intestine regulate differentiation of tissue-resident CD8 T cells responding to infection. Nature immunology, 16(4), 406-414.
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9

Isolation and Characterization of Colonic Immune Cells

Cited 1 time
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Lamina propria cells from colonic tissue were harvested as before8 (link). For intracellular cytokine staining, cells were stimulated using the eBioscience cell stimulation cocktail for 4 h at 37 °C. Cells were fixed and permeabilized using the Biolegend fixation and permeabilization buffer. The following antibodies (clones) were used for staining: CD45 (30-F11), CD19 (6D5), CD11b (M1/70), CD90.2 (53–2.1), CD3 (145–2C11), TCR-β (H57–597), CD4 (RM4–5), CD8 (53–6.7), Gr-1 (RB6–8C5), MHCII (M5/114.15.2), CD11c (N418), CD103 (2E7), IFN-γ (XMG1.2) from Biolegend and IL-22 (1H8PWSR), and IL-13 (eBio13A) from eBioscience. All samples were blocked with Fc block (TruStainfcx) and stained with a fixable live dead stain (Invitrogen). FlowJo v.10 was used to analyze flow cytometry data. Intestinal epithelial cells (IECs) were harvested by incubation at 37°C with 2mM DTT (Sigma) followed by two incubations with 5mM EDTA and then digested with Dispase (Sigma) and DNase (Sigma). IECs were sorted as PI−CD45−EpCam+ using the following antibody clones: CD45 (30-F11) and EpCam (G8.8) from Biolegend on an FACSAria II (BD Biosciences). Purity of IECs was ≥ 95%.
Neil J.A., Matsuzawa-Ishimoto Y., Kernbauer-Hölzl E., Schuster S.L., Sota S., Venzon M., Dallari S., Galvao Neto A., Hine A., Hudesman D., Loke P., Nice T.J, & Cadwell K. (2019). IFN-I and IL-22 mediate protective effects of intestinal viral infection. Nature microbiology, 4(10), 1737-1749.
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10

Immunofluorescence Analysis of SARS-CoV-2 Targets

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Tissue was collected and fixed in 4% paraformaldehyde overnight. Samples were then dehydrated in a 30% sucrose solution. Optimal cutting temperature compound–embedded 10-mm cryostat sections were blocked in 0.1 M Tris-HCl buffer with 0.3% Triton and 1% FBS before staining. Slides were stained for EpCAM (G8.8; Biolegend) with fluorochrome-labeled primary antibody. The primary antibodies rabbit anti-SARS-CoV-2 nucleocapsid (GeneTex) and rabbit anti-ACE2 (Abcam) or rabbit IgG isotype control were used and detected with secondary antibodies, donkey anti-rabbit IgG Alexa Fluor Plus 488/555 (Invitrogen). Slides were stained with DAPI (Sigma-Aldrich) and mounted with Prolong Gold Antifade reagent (Thermo Fisher Scientific). All slides were analyzed by fluorescence microscopy (BX51; Olympus) with a 10× lens. Imaging data were analyzed with Imaris 7.2 (Bitplane).
Israelow B., Song E., Mao T., Lu P., Meir A., Liu F., Alfajaro M.M., Wei J., Dong H., Homer R.J., Ring A., Wilen C.B, & Iwasaki A. (2020). Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling. The Journal of Experimental Medicine, 217(12), e20201241.
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