The protocol that was used in this study for circulating TB collection was validated and published by Vossaert et al. in 2019.3 (link) Briefly, 30–40 ml of maternal venous blood was collected. After lysis of red blood cells, trophoblasts were enriched from the remaining cells using positive immunomagnetic selection with a cocktail including anti-human HLA-G (Novus Biologicals, NB11055298B), anti-Trop2 (Novus Biologicals, NBP211982B), anti-EpCAM (BioLegend, 324216), as well as an in-house developed anti-EpCAM antibodies. The enriched cell suspension was then stained for the TB marker cytokeratin (Alexa-488, clone AE1/AE3, clone C11, BioLegend, 628608), the WBC marker CD45 clone 2D1 (VWR, 75781–326), and the nuclear stain DAPI (Thermo Fisher, D1306). Candidate TB cells were then individually scored by fluorescence microscopy using a set of criteria including nuclear shape, CD45-negative, and cytokeratin-positive staining patterns. Genotyping confirmed fetal cell identity of the isolated TBs for all cells. This was further corroborated by genome wide-copy analysis for male cells and cells with copy number changes not present in the mother’s DNA. To facilitate data analysis for this study, we defined the fetal cell concentration (FCC) for each sample as the number of TBs/100 ml of maternal blood.
Anti epcam
Manufactured by BioLegend
Sourced in United States
Anti-EpCAM is a primary antibody that specifically binds to the Epithelial Cell Adhesion Molecule (EpCAM) protein. EpCAM is a cell surface glycoprotein expressed on most epithelial cells and some cancer cells. The Anti-EpCAM antibody can be used for the detection and identification of cells expressing EpCAM.
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Lab products found in correlation
Anti cd45, by BioLegend (5 mentions)
Anti nk1, by BioLegend (3 mentions)
Anti f4 80, by BioLegend (3 mentions)
Anti ly6g, by BioLegend (3 mentions)
Anti mhc 1, by BioLegend (2 mentions)
Anti pd l1, by BioLegend (2 mentions)
Anti cd19, by BioLegend (2 mentions)
Elastase, by Roche (2 mentions)
Anti s100a4, by Abcam (2 mentions)
Anti cd31, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti sp c, by ABclonal (2 mentions)
Anti aqp 4, by Abcam (2 mentions)
Anti ssea4, by BioLegend (2 mentions)
Anti cxcr4, by BioLegend (2 mentions)
Anti cd44, by BD (2 mentions)
Anti tra 1 60, by BioLegend (2 mentions)
Anti gfap, by Merck Group (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
19 protocols using anti epcam
1
Validated Protocol for Circulating Trophoblast Isolation
2
Semi-Automated Detection of CTCs
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To validate the semi-automated microscopic approach with the NYONE® (SYNENTEC, Elmshorn, Germany), cultured HT29 human CRC cells (approximately 100 cells, achieved by repeated counting) were spiked into 8.2 mL of blood from healthy donors who gave written informed consent. These blood samples were then transferred into Vacutainer-CPT-tubes (BD) and processed according to the manufacturer´s guidelines. The enriched mononuclear cell (MNC)-fraction was later incubated and stained with Alexa488-conjugated anti-CD45 antibodies (#304017; Biolegend, San Diego, CA, USA) for the detection of leucocytes (green fluorescence) and Alexa647-conjugated anti-EGFR (#sc-120 AF647; SantaCruz, Dallas, TX, USA), anti-Her2 (#3244412; Biolegend), anti-EpCAM (#324212; Biolegend) and anti-pan-CK (#628604; Biolegend) antibodies against the CTC (red fluorescence). After a washing step, a buffer containing DAPI (#422801; Biolegend, San Diego, CA, USA) was added, and automated microscopy was performed using the NYONE® cell imager using the software package YT-software (SYNENTEC, Elmshorn, Germany) (Figure 5 A). A CTC was defined as being DAPI and Alexa-647-positive, as well as Alexa488-negative. A detailed protocol of the method is given in the Supplementary Methods .
3
Antibody Panel for Cell Characterization
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Anti-CD31 antibody was from Transduction Laboratories (Lexington, KY). Anti-AQP5, –SPB, -β-catenin antibodies were from Abcam (Cambridge, MA). Anti-β-actin monoclonal antibody was from Sigma (St. Louis, MO). Anti-VEGFR2 and LRP5 antibodies were from Cell Signaling (Danvers, MA). Anti-Tie2 monoclonal antibody was from Upstate (Lake Placid, NY). Anti-Tie2 polyclonal antibody was from Santa Cruze Biotechnology (Dallas, TX). Anti-EpCAM, -CD31, -VE-cadherin, and –CD45 antibodies were from BioLegend (San Diego, CA).
4
Comprehensive Immune Cell Profiling
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Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1 : Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).
5
Visualization and Uptake of Extracellular Vesicles
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For sEV view analysis, the isolated tissue sEVs were incubated with fluorescent antibodies of anti-EpCAM (BioLegend) and anti-CD45 (BioLegend) for 1 h at room temperature. Tissue sEVs were imaged by a Nikon A1 confocal microscope (Nikon Instruments). For uptake of sEVs, purified T lymphocyte-derived sEVs (approximately 6.4 × 109 for both normal and HT subjects) were labeled with a PKH67 green fluorescent labeling kit (Sigma‒Aldrich). Then, Nthy-ori-3–1 cells were cocultured with PKH67-labeled sEVs and visualized after 24 h [8 (link), 23 (link)].
6
Magnetic Beads for Cancer Cell Targeting
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Once the polymer beads were prepared, the next step was to functionalize them and study their interaction with EpCAM-, EGFR- and FGFR-expressing cancer cells.
The carboxylic groups of MA on the surface of the magnetic polymer beads were activated using two reagents:1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in the presence of Tween® 20 (as a dispersing agent) for 15 min at room temperature. After incubation, these reagents were removed very quickly (centrifugation at 22000 rpm for 10 min) in order to avoid bead aggregation. This activation enables reaction with primary amines to form amide bonds. The activated magnetic polymer beads were incubated with proteins (protein A or albumin) for 3 h at 4°C to allow binding of the proteins to the surface of the beads.
After protein A coating, the beads were incubated with the following selected antibodies (of mouse origin): anti-EpCAM (Biolegend), anti-EGFR (Dianova, Hamburg, Germany) and anti-FGFR (Abcam) which were efficiently attached to the surface of the beads for the cell studies.
The carboxylic groups of MA on the surface of the magnetic polymer beads were activated using two reagents:1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) in the presence of Tween® 20 (as a dispersing agent) for 15 min at room temperature. After incubation, these reagents were removed very quickly (centrifugation at 22000 rpm for 10 min) in order to avoid bead aggregation. This activation enables reaction with primary amines to form amide bonds. The activated magnetic polymer beads were incubated with proteins (protein A or albumin) for 3 h at 4°C to allow binding of the proteins to the surface of the beads.
After protein A coating, the beads were incubated with the following selected antibodies (of mouse origin): anti-EpCAM (Biolegend), anti-EGFR (Dianova, Hamburg, Germany) and anti-FGFR (Abcam) which were efficiently attached to the surface of the beads for the cell studies.
7
Flow Cytometry Analysis of Intestinal Epithelial Cells and Rotavirus Infection
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Dissociated cells were collected and stained for flow cytometry. Cells were stained with Zombie Aqua viability dye (BioLegend), Fc receptor-blocking antibody (CD16/CD32; BioLegend), anti-EpCAM (clone G8.8; BioLegend), and anti-CD45 (clone 30-F11; BioLegend). For analysis of murine rotavirus infection, cells were stained with anti-rotavirus (polyclonal; ThermoFisher, #PA1-7241) followed by goat anti-rabbit secondary (ThermoFisher). All data were analyzed using FlowJo software (BD Biosciences). Gates were set based on unstained and single-fluorophore stains. IECs were selected by gating on live, EpCAM-positive, CD45-negative cells. Gates for murine rotavirus infection were set based on naïve samples.
Where indicated, dissociated cells were enriched using MojoSort Mouse anti-APC Nanobeads (BioLegend, #480072) after flow cytometry staining for anti-EpCAM and anti-CD45 with APC fluorophores by following manufacturer protocols.
Where indicated, dissociated cells were enriched using MojoSort Mouse anti-APC Nanobeads (BioLegend, #480072) after flow cytometry staining for anti-EpCAM and anti-CD45 with APC fluorophores by following manufacturer protocols.
8
Immunofluorescence Staining of Brain Tissue
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Tissues were fixed in 4% paraformaldehyde overnight at 4 °C. Following paraffin-embedding and sectioning, slides were baked at 60 °C for 2 h, deparaffinized with successive incubations in xylene, absolute ethanol, 95% ethanol and 70% ethanol, and incubated in 1X Target Retrieval Solution (Dako) at 110 °C for 90 s. Sections were then successively incubated in blocking solution (Dako) for 1 h, primary and secondary antibodies diluted in antibody diluent solution (Dako) overnight at 4 °C and 1 h at room temperature, respectively. The following primary and secondary antibodies were used: rabbit polyclonal anti-S-100β (1:1000, DAKO), rabbit polyclonal anti-GFAP (1:500, DAKO), mouse polyclonal anti-EpCAM (1:200, Biolegend), anti-rabbit-Cy3 (1:500, Jackson) and anti-mouse-Alexa Fluor 488 (1:500, Invitrogen). Stained sections were imaged using an OLYMPUS BX51 microscope (objectives 20X FN 26.5, 40X FN 26.5) and with an OLYMPUS IX83 inverted microscope (objectives 20X FN 22, 40X FN 22).
9
Isolation of Murine Lung Cell Subsets
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BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through a 40 μm nylon mesh and then sorted by FACS. Anti-CD45 (BioLegend, 157607, 1:200 dilution) and anti-CD3 (BioLegend, 100209, 1:200 dilution) antibodies were used for the isolation of T cells. Anti-CD45 and anti-CD19 (BioLegend, 152407, 1:200 dilution) antibodi were used for isolation of B cells. Anti-CD45 and anti-NK-1.1 (BioLegend, 108703, 1:200 dilution) antibody were used for isolation of NK cells. Anti-CD45 and anti-F4/80 (BioLegend, 123105, 1:200 dilution) antibodies were used for the isolation of macrophages. Anti-CD45 and anti-Ly6G (BioLegend, 127627, 1:200 dilution) antibodies were used for the isolation of neutrophils. Anti-EpCAM (BioLegend, 324215, 1:200 dilution) and anti-SP-C (abclonal, A1835, 1:500 dilution) antibodies were used for the isolation of alveolar epithelial cells. anti-CD31 (BioLegend, 102409, 1:200 dilution) antibody was used for the isolation of endothelial cells. Anti-S100A4 (abcam, ab197896, 1:500 dilution) antibody was used for the isolation of fibroblasts.
10
Cell Sorting and Molecular Analysis of HCC
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For CD133+ and EpCAM+ cell sorting, primary HCC patients’ cells and hepatoma cells were incubated with the primary anti-CD133 (cat. no. 372806; BioLegend, San Diego, CA, USA) or anti-EpCAM (cat. no. ab8666; Abcam, USA) for 30 min at room temperature. The cells were then subjected to flow cytometry using a MoFlo XDP cell sorter from Beckman Coulter (Indianapolis, IN, USA), according to the manufacturer’s instructions. The sorted cells from three independent experiments were subjected to real-time PCR assay.
miR-552 mimic or miR-552 sponge and control hepatoma cells were incubated with the primary anti-EpCAM for 30 min at room temperature. The flow cytometry analysis was performed using a MoFlo XDP from Beckman Coulter, according to the manufacturer’s instructions.
miR-552 mimic or miR-552 sponge and control hepatoma cells were incubated with the primary anti-EpCAM for 30 min at room temperature. The flow cytometry analysis was performed using a MoFlo XDP from Beckman Coulter, according to the manufacturer’s instructions.
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