As described previously (16 (link)), murine CAFs were isolated from lung tissues at necropsy by immediately perfusing tissues with 2% fetal bovine serum in Hank’s buffered salt solution (FBS-HBSS) and dispersing them into single cell suspension by immersion in 3 mg/mL of collagenase and Dispase®II (Roche) on a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) using the lung tissue dissociation programs (Lung_01 and Lung_02). Dispersed cells were centrifuged, washed with FBS-HBSS, and subjected to red blood cell lysis by adding RBC Buffer (BioLegend). The remaining cells were centrifuged, washed, filtered (70 µm and 40 µm), counted (Countess™, Invitrogen), and mixed twice with antibody-conjugated magnetic beads (Dynal-Magnetic beads, Invitrogen) on a rotator, each time for 45 min at 4°C, to first deplete leukocytes (anti-CD45 and anti-CD68, Abcam), endothelial cells (anti-CD31, BD Pharmingen), and epithelial cells (anti-EPCAM, Pharmingen) and then to isolate fibroblasts (anti-Thy-1, BD Pharmingen) from the supernatants. Fibroblasts were eluted off the anti-Thy-1-conjugated beads by incubation in FBS-HBSS, 0.5% BSA, and 2mM EDTA, centrifuged, washed, and cultured in RPMI1640 containing 10% FBS and 100 mg/100U penicillin-streptomycin (GIBCO).
Anti epcam
Manufactured by BD
Sourced in United States
Anti-EPCAM is a laboratory reagent used in the detection and analysis of the epithelial cell adhesion molecule (EPCAM) protein. It is a monoclonal antibody that specifically binds to the EPCAM protein, which is commonly expressed on the surface of epithelial cells. Anti-EPCAM can be used in various laboratory techniques, such as flow cytometry and immunohistochemistry, to identify and quantify EPCAM-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Dynal magnetic bead, by Thermo Fisher Scientific (1 mentions)
Anti cd45, by Abcam (1 mentions)
Rbc buffer, by BioLegend (1 mentions)
Anti thy1, by BD (1 mentions)
Anti cd68, by Abcam (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dispase 2, by Roche (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Anti cd31, by BD (1 mentions)
Anti cxcr4, by BD (1 mentions)
Anti cd24, by BD (1 mentions)
Anti cd44, by BD (1 mentions)
Fix and perm reagent, by BD (1 mentions)
Anti oct3 4, by BD (1 mentions)
Anti foxa1 2, by BD (1 mentions)
Anti pdx1, by BD (1 mentions)
Anti cd338, by R&D Systems (1 mentions)
Anti nanog, by BD (1 mentions)
Anti ck19, by Abcam (1 mentions)
Anti ck7, by Abcam (1 mentions)
4 protocols using anti epcam
1
Isolation of Murine Cancer-Associated Fibroblasts
2
Immunophenotyping of Stem-like Cancer Cells
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Immunophenotyping of 82.3 cells was performed by flow cytometric analysis. Cells were washed in 1× PBS containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MO, USA) and 0.01% sodium azide. For intracellular markers, cells were permeabilized with fix and perm reagent (BD Italia) following the manufacturer’s instructions. The following antibodies were used: FITC (Fluorescein isothiocyanate) conjugated mouse mAbs anti-CK7 and anti-CK19 (Abcam, Cambridge, UK) and anti-CD44 (BD Bioscience, San Jose, CA, USA), APC-(allophycocyanin) conjugated mouse mAbs anti-EPCAM and anti-AFP (BD Bioscience Europe), anti-CD34 and anti-CD133 (Miltenyi Biotec S.r.l., Bologna, Italy), PE (phycoerytrin) conjugated mAbs anti-CD24, anti-CXCR4, anti-Oct3/4, anti-FOXA1/2, anti-PDX1, (all from BD), anti-CD338 (R&D Systems, Inc., Minneapolis, MN, USA), PerCP-Cy 5.5 conjugated mAbs anti-SOX2/17, Alexa Fluor 647 conjugated mAbs anti-Nanog, anti-Stro1, and Alexa Fluor 488 conjugated mAbs anti-PAX6 (BD Bioscience Europe).
3
Characterization of Tumor-Infiltrating Immune Cells
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Mice (n = 3) were IP inoculated with 2 × 105 CT-26-Luc cells on Day 4. On Day 0, mice were IP injected with 15 mg/kg luciferin and then imaged using the Lumina IVIS system (PerkinElmer, Waltham, MA, USA) to confirm the presence of tumors in the peritoneal cavity. Mice then received 6 mg/kg OXA via IP injection. The following morning, each mouse received an IP dose of DiI-labeled DSTAP-liposomes at 10 mg lipid/kg. After 1 h, mice were sacrificed, and IP was instilled with 5 mL of ice-cold PBS for PF collection. The PF was centrifuged to obtain IP cells, which were resuspended in a staining buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide), counted, dispensed into a microtube, and stained with various antibodies (anti-CD45, anti-CD11b, anti-CD11c, anti-F4/80, anti-CD335, anti-CD3, anti-CD8, and anti-EPCAM, BD Life Sciences, Franklin Lakes, NJ, USA) for 30 min on ice in the dark. The cells were centrifuged, resuspended in the fluorescence-activated cell-sorting buffer containing 1 mg/mL propidium iodide, and analyzed by Cytoflex LX (Beckman Coulter, Brea, CA, USA).
4
Multiparametric Flow Cytometry Profiling
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The phenotype of solid tumor and cultured cells was measured by flow cytometry analysis after washing with a buffer containing 2% BSA and staining with antimouse mAbs. The following antibodies were used: antiCD117/cKit (#11-1171-85), antiCD11b (#17-0112-83), antiCD49f (#12-0495-82; all from eBioscience), antiFceRI (#60-1172-U100, Tonbo Biosciences), and anti-EpCAM (#563478; BD Biosciences). Samples were analyzed by using FACSCalibur (BD Biosciences) and LSRII Fortessa (BD Biosciences) instruments and analyzed with FlowJo software.
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