For the extraction of colonic epithelial cells, normal or Salmonella-infected colon tissues of C56/B6 mice were collected from the mouse model (5 mice per group). After three washes with sterile PBS, the colonic lumens were injected and filled with trypsin (Thermo Fisher Scientific, 25,200,072). The two ends of the colon lumen were sealed with surgical thread ties and incubated in a cell incubator for 1 h at 37°C. After digestion, these epithelial cells in the colonic lumen could be pipetted and ejected. Then, a 10% FBS was added to the cell suspensions, and the colonic epithelial cells and non-epithelial cell colon tissues were obtained. After centrifugation and two washes with ice-cold PBS, these newly isolated epithelial cells were cultured in DMEM (10% FBS) for approximately 30–60 min. The suspended epithelial cells in the culture medium were then collected for further determination. The purity of epithelial cells was determined by flow cytometry using EpCAM antibodies (Biolegend, 118,207), a marker of epithelial cells.
Epcam antibody
Manufactured by BioLegend
Sourced in United States
The EpCAM antibody is a laboratory reagent used for the identification and detection of EpCAM (Epithelial Cell Adhesion Molecule), a cell surface glycoprotein expressed on epithelial cells. This antibody can be used in various analytical techniques, such as flow cytometry and immunohistochemistry, to study the expression and distribution of EpCAM in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg2b secondary antibody, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Collagenase hyaluronidase, by STEMCELL (1 mentions)
C12fdg, by Thermo Fisher Scientific (1 mentions)
Bafilomycin a1, by Enzo Life Sciences (1 mentions)
Cd24 antibody, by BioLegend (1 mentions)
8 protocols using epcam antibody
1
Isolation of Colonic Epithelial Cells
2
Flow Cytometry Analysis of EpCAM Expression
3
CTC Identification via Immunofluorescence Staining
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Two slides from patient UM-001 were stained with pan-cytokeratin (CK) and CD45 antibodies as described above with DAPI24 (link) complemented with a monoclonal EpCAM antibody (1:250, 324202, Biolegend, San Diego, CA; Alexa Fluor® 488 goat anti-mouse IgG2b secondary antibody, 1:500, A21141, Invitrogen, Carlsbad, CA) at Epic Sciences. CTCs were identified with standard image analysis protocols25 (link)–35 (link).
4
Isolation of Murine Pancreatic Tumor Cells
5
Isolating Luminal Prostate Cells for ATAC-seq and RNA-seq
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Intraperitoneal injection of TAM was administered in 8-week-old mouse. Two weeks after TAM treatment, mouse prostate was digested 1 hour with collagenase/hyaluronidase (STEMCELL Technologies, no. 07912) and then 30 min with TrypLE Express Enzyme (Thermo Fisher Scientific, no. 12605028) at 37°C to isolate single prostate cells. The prostate cells were stained with phycoerythrin/cyanine 7–conjugated anti-mouse CD326 (EPCAM) antibody (BioLegend, 118216), and then, CD326 and EYFP double-positive cells were sorted out by flow cytometry, which are luminal cells mainly from anterior prostate and dorsal prostate. The mRNA or genomic DNA was extracted from these double-positive cells and then was used for ATAC-seq and RNA-seq analysis.
6
Identifying Phagocytic Macrophages in Tumor Samples
7
Flow Cytometric Analysis of SA-β-Galactosidase
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Flow cytometry-based detection of SA-β-galactosidase was performed as described previously [28 (link)]. Briefly, pmATII cells from PBS- and bleomycin-treated animals were incubated with bafilomycin A1 (100 nM; Enzo Life Sciences, Farmingdale, NY, USA) and C12FDG (20 nM; Life Technologies, Carlsbad, CA, USA) for 1 and 2 h, respectively, directly after isolation or at day 2 of culture. Cells were washed once and stained for allophycocyanin-conjugated epithelial cell adhesion molecule (EpCAM) antibody (118214; BioLegend, San Diego, CA, USA) for 20 min at room temperature, washed once and analysed using a fluorescence-activated cell sorter (LSRII; BD Bioscience, San Jose, CA, USA). Additional information can be found in the online supplementary material.
8
Identification of Liver Cancer Stem Cells
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Single-cell suspensions (Hep3B SP and Huh7 SP) were prepared and incubated with CD24 antibody (BioLegend Cat#311,106, RRID: AB_314855), EPCAM antibody (BioLegend Cat#324,212, RRID: AB_756086), and CD133 antibody (Biolegend, #S16016B, RRID: AB_2734479) for 30 min in 4 °C, followed by flow cytometry analysis. LCSCs (LCSC1 and LCSC2) were prepared and incubated with ALDH antibody (STEMCELL, Cat#01700) for 30 min in 37 °C, followed by flow cytometry analysis.
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