The stem cells were dissociated with trypsin, washed with cold PBS, and then separately stained with immunoglobulin G (IgG) or the following monoclonal antibodies conjugated to PE, PerCP, APC, or FITC: Epcam-PE, CD44-FITC, CD29-FITC, CD49f-FITC, CD73-FITC, CD105-APC, CD90-FITC, CD34-PerCP, CD31-FITC, CD45-FITC, and HLA-DR-FITC (all from eBioscience, USA). Upon being washed with PBS, the labeled cells were resuspended, and at least 105 events were acquired by using a BD Accuri™ C6 flow cytometer (BD, USA).
Epcam pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Epcam-PE is a fluorochrome-conjugated monoclonal antibody that binds to the epithelial cell adhesion molecule (EpCAM) protein expressed on the surface of epithelial cells. It can be used for the detection and quantification of EpCAM-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 apc, by Thermo Fisher Scientific (2 mentions)
Cd31 fitc, by BioLegend (2 mentions)
Pe cy7 ter 119, by BioLegend (2 mentions)
Live dead fixable violet dead cell stain, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Cd31 fitc, by Thermo Fisher Scientific (1 mentions)
Cd44 fitc, by Thermo Fisher Scientific (1 mentions)
Cd29 fitc, by Thermo Fisher Scientific (1 mentions)
Cd90 fitc, by Thermo Fisher Scientific (1 mentions)
Cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Hla dr fitc, by Thermo Fisher Scientific (1 mentions)
Cd49f fitc, by Thermo Fisher Scientific (1 mentions)
Cd34 percp, by Thermo Fisher Scientific (1 mentions)
Cd105 apc, by Thermo Fisher Scientific (1 mentions)
Cd73 fitc, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Cd49f apc, by Thermo Fisher Scientific (1 mentions)
Cd24 pe, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Tween 20, by Merck Group (1 mentions)
6 protocols using epcam pe
1
Phenotypic Characterization of Stem Cells
2
Characterizing Cell Surface Markers in DEAR1-KD Clones
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Control clones and DEAR1-KD 76N-E6 clones were grown in 2D culture and stained for FACS using EpCAM-PE (eBioscience, 12-9326-41), CD49f-APC (eBioscience, 17-0495-80), CD24-PE (BD Biosciences, 555428), and CD44-FITC (BD Biosciences, 555478) according to the manufacturer’s Staining Cell Surface Antigens for Flow Cytometry protocol. Plated cells were trypsinized, counted, and resuspended in Flow Cytometry Staining Buffer for a final concentration of 1 × 107 cells/mL. 50 µL of the cell suspension was incubated with primary antibody so that the final volume was 100 µL per sample. The cells incubated for 30 min in the dark at 4 °C, washed, and analyzed. For these experiments, 2 stable control vector (CshR) clones and 2 DEAR1-KD (DshR) stable clones were used experiments were performed in triplicate.
3
Cell Cycle Regulation Analysis
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For cell cycle regulation assessment, cells were washed in 1× PBS and 10 μM BrdU-APC was added to cells in a dark environment. Plates with BrdU were then incubated at 37 °C for 4 h. Cells were then washed twice with ice cold PBS, trypsinized with 0.25% trypsin dissociation reagent and neutralized for counting. Nuclei preparation and staining was performed by adding 0.08% Pepsin and 2 N HCl and IFA/0.5%, Tween20 (Sigma). Samples were incubated in the dark and 100 μl anti-BrdU-APC was added while samples were incubated on ice. 5 μg/ml of propidium iodide (PI) was added to cells and incubated on ice for 15 min. Flow cytometry was carried out on BD FACS Calibur (BD biosciences, San Jose, CA, USA). Data was analyzed using FlowJo v10 (Flowjo LLC, Ashland, OR, USA). Cell surface staining was performed on single cell suspension. EpCAM-PE (Life technologies, VU-1D9) and CD49f-APC (FAB13501A) (R&D Biosystems, Minneapolis, Minnesota, USA) was added to the cell suspension and incubated in the dark for 40-60 min. Cells were then washed and centrifuged at 400 g for 5 min at room temperature and subsequently vortexed to dissociate pellet. Stained cells were re-suspended in staining buffer and flow cytometry performed on BD Calibur flow cytometer (BD biosciences).
4
Isolation of rPGCLCs by FACS
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The rPGCLCs at D1, D2, and D4 were dissociated using 0.05% trypsin, while D8 PGCLCs were dissociated using 0.05% trypsin and Collagenase IV. Cells were washed with MEF media and then re-suspended in FACS buffer. Dissociated cells were incubated with anti-ITGA6-BV421 (BioLegend 313624; 1:50) and EPCAM-PE (Life Technologies A15782; 1:50) antibodies. Double positive cells were collected using an ARIA-H Fluorescence Activated Cell Sorter. Cells were sorted into RLT buffer and stored at −20 °C until ready to isolate RNA. Sorts of rPGCLCs were performed on at least 2 replicates for each group. Cytometry analysis was performed using FlowJo™ version 10.
5
Isolation and FACS-Sorting of Intestinal Epithelial Cells
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ECs were isolated as previously described (Haber et al., 2017 (link)), with modifications. Briefly, the ileal, cecal, and colonic tissues of VB12- and PBS-gavaged mice were dissected and washed with cold PBS. The tissues were opened longitudinally and cut into small fragments (2–3 cm in length), followed by incubation with 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 8–10 min. Isolated crypts were washed once with cold PBS and dissociated with TrypLE Express (Invitrogen) for 10 min at room temperature. The single-cell suspension was passed through a 70-μm cell strainer and used for FACS. To FACS-sort the cells, cell suspensions were labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (catalog 17-0451-83; Invitrogen), FITC-CD31 (catalog 102406; BioLegend), PE/Cy7-TER-119 (catalog 116222; BioLegend), and PE-EpCAM (catalog 12-5791-83; Invitrogen). Dead cells were excluded using LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen). CD45− CD31− TER-119− EpCAM+ ECs were sorted using a SONY SH800S Cell Sorter or a CytoFLEX SRT Cell Sorter.
6
Isolation of Intestinal Epithelial Cells
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The ileum (similar length as colon moving proximal from the ileocecal junction), cecum and colon were dissected from mice after MCAO or sham surgery. After washing with cold PBS, the tissues were opened longitudinally and cut into small fragments (2–3 cm in length), followed by incubation with 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 8–10 min. Isolated crypts were washed once with cold PBS and dissociated using TrypLE Express (Invitrogen) for 10 min at room temperature. Cell suspensions were passed through a 70-μm cell strainer and then labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (Invitrogen, catalog 17-0451-83), FITC-CD31 (BioLegend, catalog 102406), PE/Cy7-TER-119 (BioLegend, catalog 116222), and PE-EpCAM (Invitrogen, catalog 12-5791-83). Dead cells were excluded using LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen). CD45− CD31− TER-119- EpCAM+ ECs were sorted using a SONY SH800S Cell Sorter.
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