JC1 staining was performed on the above single-cell isolations with flow cytometry using the JC-1 (5,5″,6,6″-tetrachloro-1,1″,3,3″-tetraethylbenzimidazolylcarbocyanine iodide, Molecular Probes, Inc. Eugene, OR) reagent according to the manufacturer’s instructions. In brief, JC-1 dye was added at 1 mcM to washed cells, and incubated for 20 min at 37 °C, 5% CO2. Cells were washed and CD45 APC-Cy7 (BD Bioscience, Franklin Lakes, NJ) and EpCAM APC (BioLegend, San Diego, CA) antibodies were added for an additional 30 min at room temperature. Cells were washed, acquired on a Canto flow cytometer, and data were analyzed using DeNovo software. The MMP was calculated as the ratio of PE-MFI/FITC-MFI in EpCAM+ and CD45+ cells. Representative data are shown in Supplemental Fig. 1 . As a positive control for the specificity of the assay we used 50 mcM of CCCP (carbonyl cyanide 3-chlorophenylhydrazone) to depolarize the MMP measured using the JC-1 dye.
Epcam apc
Manufactured by BioLegend
Sourced in United States
EpCAM-APC is a monoclonal antibody that specifically binds to the Epithelial Cell Adhesion Molecule (EpCAM) protein. EpCAM is a cell surface glycoprotein that is expressed on epithelial cells and their derived tumors. The APC fluorescent dye is conjugated to the antibody, allowing for detection and quantification of EpCAM-expressing cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
Cd24 pe, by BioLegend (2 mentions)
Cd45 pe cy7, by BioLegend (2 mentions)
Cd31 pe cy7, by BioLegend (2 mentions)
Cd31 pe, by BioLegend (2 mentions)
Facs influx instrument, by BD (2 mentions)
Cd45 apc cy7, by BD (1 mentions)
Streptavidin fitc, by BD (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
H3k27me3, by Merck Group (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
P19arf, by Abcam (1 mentions)
Krt20, by Agilent Technologies (1 mentions)
Ncl l ki67 mm1, by Abcam (1 mentions)
Alexa 488, by Jackson ImmunoResearch (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Cd45 bv421, by BioLegend (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Pbs buffer, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
19 protocols using epcam apc
1
Mitochondrial Membrane Potential Assay
2
Immunohistochemical Profiling of Skin Samples
3
Cre-Mediated Lung Cell Isolation
4
Isolation of Murine Lung Cell Types
5
Multimarker Phenotyping of HGSOC Cells
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Cryopreserved or fresh HGSOC single-cell suspensions were washed and resuspended at ≤107 cells/ml in FC buffer and incubated with 10 µg/ml mouse IgG in FC buffer on ice for 10 min, followed by incubation with the following primary antibodies: CD45-PECy7 (BioLegend; 1:200), CD31-PECy7 (BioLegend; 1:200), CD49e-PE (BD Biosciences; 1:100), EpCAM-APC (BioLegend; 1:100). For experiments including CD133, an unconjugated mouse anti-human CD133 antibody was used (Miltenyi Biotec; 1:20), and for experiments including FAP, an unconjugated mouse anti-FAP antibody was used (R&D Systems; 1:50). Cells were incubated on ice for 15 min, washed and resuspended in FC buffer with goat-anti-mouse Alexa488 (Invitrogen; 1:400) for an additional 15 min, and then washed and stained with the remaining directly conjugated antibodies as described above. Fluorescence-minus-one controls were generated for each antibody and used as gating controls. Single-color stained compensation beads (BD Biosciences) were used as compensation controls. Cells were analyzed on a BD LSR II flow cytometer or sorted using a BD FACS Aria.
6
Immunophenotyping of PDBCCs by Flow Cytometry
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Single cells dissociated from spheroids or monolayers with TrypLE™ (Thermo Fischer, Waltham, MA, USA) were stained using concentrations of fluorochrome-conjugated monoclonal antibodies recommended by the manufacturer for 20 min at room temperature in the dark. Antibodies for EpCAM-APC (BioLegends, San Diego, California, USA), CD24-FTIC (BioLegends, San Diego, California, USA), CD44-PE (BioLegends, San Diego, California, USA), CD49f-FITC (BioLegends, San Diego, California, USA), and fibroblast-marker-FITC (BioLegends, San Diego, California, USA) were used. Expression of EpCAM, CD24, CD44, CD49f, and fibroblast-marker on PDBCCs was analyzed by multicolor flow cytometry on a FACS Canto II flow cytometer (BD Biosciences, Becton Drive Franklin Lakes, NJ, USA). The gating strategy was provided in Supplementary Fig. 1 .
7
Lung Cell Isolation and FACS Analysis
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For FACS analysis, lungs were digested in collagenase (1mg/ml collagenase in 3ml DPBS + 0.2g/L glucose per lung) at 37 °C for 60 min while shaking at 165 rpm, followed by red blood cell lysis with 0.64 % ammonium chloride at 37 °C for 3 min [31 (link)]. Cells were resuspended in blocking solution (anti-FcR and Rat IgG) and incubated on ice for 10 min. Cells were stained with conjugated antibodies CD31-PECy7, CD45-PECy7, EpCAM-APC, CD104-FITC and CD24-PE (Biolegend) as described previously [32 (link)]. Cells were then washed and resuspended in PI solution. Cells were sorted on an ARIA II (Beckton Dickinson).
8
Enrichment and Lysis of Thymic Epithelial Cells
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Single-cell suspensions from two or three thymi following Percoll enrichment for TECs (see above) were further enriched for TECs by staining for EpCAM-APC (118214; Biolegend), followed by positive selection using anti-APC microbeads (130–090-855; Miltenyi Biotec). The enriched cells were washed in MACS buffer (340 g, 5 min, 4°C), and the pellet was lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 5 mM EDTA, 1% IGEPAL CA-360, 0.5% sodium deoxycholate, and 0.1% SDS) complemented with complete proteinase inhibitors (539134; Calbiochem).
Organs were surgically removed from NSG mice, and 100 mg of the tissue was lysed in 1 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% IGEPAL CA-360) supplemented with complete protease inhibitors and were processed with a tissue homogenizer. Homogeneous lysates were spun at 12,000 g for 20 min at 4°C. The supernatant was collected and supplemented with 4× sample buffer and boiled for 5 min at 96°C.
Organs were surgically removed from NSG mice, and 100 mg of the tissue was lysed in 1 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% IGEPAL CA-360) supplemented with complete protease inhibitors and were processed with a tissue homogenizer. Homogeneous lysates were spun at 12,000 g for 20 min at 4°C. The supernatant was collected and supplemented with 4× sample buffer and boiled for 5 min at 96°C.
9
Detection of Spiked Breast Cancer Cells
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Aliquots (0.5 ml) from the previously prepared 15 ml Falcon tubes, containing ~ 250,000 cells from a cancer-cell line spiked into normal whole blood, were analyzed by flow cytometry. Red blood cells were lysed using ammonium chloride, and samples were then stained with the antibodies CD45-PC7 (25-0459-42, eBioscience, Wien, Austria) and CD31-RPE (MCA1738PE, Abd Serotec, Segrate, Italy), which were used to exclude CD45+ cells and endothelial cells, respectively, during analysis of the data. The antibody EPCAM-APC (324208, Biolegend, CA, USA) was used to identify cancer cells of epithelial origin and CD227-FITC (MCA1742F, Abd Serotec) was used to further identify breast-cancer cells expressing mucin-1. Flow cytometry was performed on a Beckman Coulter FC500 flow cytometer equipped with a 488 nm blue laser and a 633 nm red, air-cooled laser. Each experiment was performed in triplicate.
10
Quantifying G-CSF in Murine Sepsis
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Mice were injected intraperitoneally with 50 μg of LPS and killed 2 or 3 h postinfection as detailed in the figure legends. Human (Sigma #RAB0103-1KT) and mouse (Sigma #RAB0104-1KT) G-CSF levels were quantified by ELISA in serum, bone marrow aspirates, lung homogenates (0.1 g), sorted endothelial cells, and sorted epithelial cells. Single-cell suspensions from lung and liver were stained with CD31-PE (BioLegend) and EPCAM-APC (BioLegend). Endothelial cells and epithelial cells were sorted using Sony SH800 Cell Sorter (100,000 cells). All tissue samples and cells were lysed in 1× RIPA buffer to allow release of G-CSF from cells. These lysates were spun at 10,000 × g for 10 min. Supernatant was used in G-CSF protein quantification at least in duplicates. ELISA kits used could detect G-CSF levels as low as 1 pg/mL.
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