Epcam fitc

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EpCAM-FITC is a fluorescent-labeled antibody that binds to the Epithelial Cell Adhesion Molecule (EpCAM) antigen. EpCAM is a transmembrane glycoprotein expressed on the surface of epithelial cells and some types of cancer cells.

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EpCAM (75 citations) EpCAM-FITC (9C4; (4 citations) FITC‐conjugated anti‐EpCAM (3 citations) Anti-EpCAM-FITC (3 citations) EpCAM-FITC antibody (2 citations) FITC-conjugated anti-mouse CD326 (EpCAM) mAb (2 citations) FITC anti-human CD326 (EpCAM) antibody (2 citations) FITC anti-mouse CD326 (EpCAM) (2 citations) FITC-conjugated EpCAM (2 citations) FITC anti-human CD326 (EpCAM) (2 citations)

9 protocols using epcam fitc

Quantifying Lung Cell Populations

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14 d after elastase instillation, 25 µg of VE-Cadherin Alexa Fluor 647 (Biolegend; BV13) was injected retro-orbitally under anesthesia 8 min before sacrifice. Lungs were minced and incubated with Collagenase A (2.5 mg/ml; Sigma-Aldrich), Dispase II (1.0 mg/ml; Sigma-Aldrich), and DNase (80 U/ml; Sigma-Aldrich) at 37°C for 30 min. Single cells were stained with CD45-BV421 (Biolegend; 30-F11), CD31-PE/Cy7 (Biolegend; MEC13.3), EpCAM-FITC (Biolegend; G8.8), and DAPI and quantified with BD FACS aria II. ECs are defined as CD45⁻VEcadherin⁺CD31⁺cells, whereas EpCAM⁺ epithelial cells are defined as CD45⁻VEcadherin⁻CD31⁻EpCAM⁺ cells. Proportion is defined as a ratio of either endothelial or epithelial cell count to total cells counted in lung. For analysis of EC compartment after intravenous delivery of lung ECs, VEcadherin (intravital labeling) cells were quantified with FACS analysis 28 d after elastase instillation.

Hisata S., Racanelli A.C., Kermani P., Schreiner R., Houghton S., Palikuqi B., Kunar B., Zhou A., McConn K., Capili A., Redmond D., Nolan D.J., Ginsberg M., Ding B.S., Martinez F.J., Scandura J.M., Cloonan S.M., Rafii S, & Choi A.M. (2021). Reversal of emphysema by restoration of pulmonary endothelial cells. The Journal of Experimental Medicine, 218(8), e20200938.

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Mammary Gland Cell Sorting and Culture

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Thoracic and inguinal mammary glands were dissected from 9-week-old p38α^(lox/lox);MMTV-Cre and MMTV-Cre virgin females or 6-week-old p38α^(lox/lox);MMTV-Cre;PyMT and MMTV-Cre;PyMT female mice. Cell suspensions were prepared as previously described (Prater et al., 2013 (link)). FACS sorting was performed with a FACSAria sorter (BD Bioscience). Data were analyzed with the FlowJo software package. The antibodies used for FACS were from Miltenyi Biotec: Epcam-APC (#130102234), Epcam-FITC (#130102214), CD49f-APC (#130097250), CD49f-PE (#130097246), BP1-biotin (#130101844), CD31-biotin (#130101955), CD45-biotin (#130101952), and Ter119-biotin (#130101882); and from Biolegend: CD61-AF480 (#104311), Sca1-PECy7 (#108114), CD49b-PE (#103506), and Streptavidin-APCCy7 (#405208). FACS gating was based on single color staining and fluorescence-minus-one controls, and was checked with isotype controls (Prater et al., 2013 (link)). Gating strategies were as described previously (Prater et al., 2013 (link), Shehata et al., 2012 (link)). For colony-formation assays, freshly sorted cells were embedded in Matrigel (BD Pharmingen) as described previously (Shackleton et al., 2006 (link)).

del Barco Barrantes I., Stephan-Otto Attolini C., Slobodnyuk K., Igea A., Gregorio S., Gawrzak S., Gomis R.R, & Nebreda A.R. (2017). Regulation of Mammary Luminal Cell Fate and Tumorigenesis by p38α. Stem Cell Reports, 10(1), 257-271.

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Immunofluorescent Tissue Staining Protocol

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Intestinal sections and lungs were harvested and snap-frozen in OCT as above. The cryo-sections (6 μm) were fixed with 10% neutral buffered formalin solution (Sigma) for 10 min at room temperature (RT), rehydrated in PBS, then blocked with 10% normal goat (Vector Labs) or donkey (Abcam) serum with 0.3% Triton X-100 (Sigma) for 20 min at room temperature (RT). The tissues were incubated with primary antibodies (EpCAM-FITC, Biolegend; rabbit anti-mouse ZO-1, Abcam; rabbit polyclonal anti-MUC2, Abcam) overnight at 4°C. The secondary antibodies (goat anti-rabbit IgG-Alexa Fluor 555, Thermo Fisher, donkey anti-rabbit IgG-Alexa Fluor 488, Thermo Fisher) were added the following day after X2 PBS washes and incubated for 1 hour at RT. The slides were finally mounted in Prolong Antifade Mountant with DAPI (Thermo Fisher) or Vectashield with DAPI (Vector Labs), imaged on a Zeiss fluorescence microscope using AxioVision or Zen lite (Blue edition) software and analyzed using Fiji (ImageJ) for mean fluorescence intensity.

Matei D.E., Menon M., Alber D.G., Smith A.M., Nedjat-Shokouhi B., Fasano A., Magill L., Duhlin A., Bitoun S., Gleizes A., Hacein-Bey-Abina S., Manson J.J., Rosser E.C., Klein N., Blair P.A, & Mauri C. (2021). Intestinal barrier dysfunction plays an integral role in arthritis pathology and can be targeted to ameliorate disease. Med (New York, N.y.), 2(7), 864-883.e9.

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Isolation and Characterization of Lung Cells

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Lungs were minced using a razor blade and incubated in dissociation solution containing DNAse (40 U/ ml, Roche) and Collagenase IV (1,000 U/ml, Worthington) rotating at 37°C for 1 hour. Following red blood cell lysis, cells were blocked for 15 minutes at 4°C and stained with the following primary antibodies for 25 minutes at 4°C: CD31-APC (BD Pharmingen), CD45-PE-Cy7 (BD Pharmingen), Epcam-FITC (Biolegend). DAPI (Invitrogen) was used to exclude dead cells. FACS sorting was carried out on a BD FACSAria (BD Biosciences) prior to RNA sequencing, qRT-PCR, or immunoblot analyses.

Dimitrova N., Gocheva V., Bhutkar A., Resnick R., Jong R.M., Miller K.M., Bendor J, & Jacks T. (2015). Stromal expression of miR-143/145 promotes neoangiogenesis in lung cancer development. Cancer discovery, 6(2), 188-201.

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Multiparameter Flow Cytometry of Immune Cells

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Single cell suspensions from mouse experiments were stained with Live/dead Aqua (Molecular Probes, Inc.), CD4-PerCP, CD8-Pacific Blue, CD69-BV605, CD62E-PE, CD62E-BV421, CD62P-Alexa647, CD162-AlexaFlour647, HECA454-PE (BD Bioscience), CD25-APC, CD31-BV605, CD105-Pacblue, I/A-I/E-BV421, CD45-APCCy7, EpCAM-APC-Cy7, Podoplanin-PE, CD31-PECy7, EpCAM-FITC, CD64-BV711 (Biolegend), CD11c-PECy5.5 (Invitrogen) CXCR3-PECy7, CD3-Alexa700 and Ly6C-efluor450 (eBioscience). For subsequent detection of CXCL10 mRNA Primeflow^® RNA Assay (eBioscience) was used accordingly to manufacture protocol. Samples were acquired on an LSR-II flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Single cell suspension isolated from human tumors and unaffected colon tissue were stained with Live/dead Aqua (Molecular Probes, Inc.), CD31-Alexa700, (Biolegend), CD4-PerCP, CD8-BV711, CD105-APC, CD14-Alexa700, CD19-APCH7 and CD19-PE-CF594 (BD bioscience) followed by permeabilization with Fix & Perm kit (ADG Bio research GMBH) and staining with CXCL9-FITC (R&D) and CXCL10-PE (Biolegend), flow cytometry analyses were performed as above.

Akeus P., Szeponik L., Ahlmanner F., Sundström P., Alsén S., Gustavsson B., Sparwasser T., Raghavan S, & Quiding-Järbrink M. (2018). Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice. Cancer Immunology, Immunotherapy, 67(7), 1067-1077.

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Immunophenotyping of RMC219 and RMC2C cells

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Wildtype RMC219 and RMC2C cells were harvested and 1 × 10⁶ cells were resuspended in buffer A (PBS 1X, EDTA 2 mM, inactivated FCS 1%) and 5uL of Human TruStain FcX (Biolegend, 422301) was added for 10 min at room temperature. Following blocking, cells were stained for 1 h with 5 µL of conjugated EPCAM-FITC (Biolegend, 324203) and conjugated CD44-PE (Biolegend, 103023). Following two PBS washes, cells were resuspended in buffer A before flow cytometry on a LSRII Fortessa (BD Biosciences) and analysis using Flowjo v6.8.

Vokshi B.H., Davidson G., Tawanaie Pour Sedehi N., Helleux A., Rippinger M., Haller A.R., Gantzer J., Thouvenin J., Baltzinger P., Bouarich R., Manriquez V., Zaidi S., Rao P., Msaouel P., Su X., Lang H., Tricard T., Lindner V., Surdez D., Kurtz J.E., Bourdeaut F., Tannir N.M., Davidson I, & Malouf G.G. (2023). SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance. Nature Communications, 14, 3034.

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Isolation and Culture of Mammary Epithelial Cells

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Mouse mammary glands were dissected and analyzed as previously described^{16 (link)}. Briefly, the glands were digested with collagenase/hyaluronidase for 2 hours followed by ACK. Then a 2 minutes Trypsin and DNAse/Dispase digestion was performed. The cells were then stained with the following antibodies: EpCAM-FITC, CD49f-APC, CD24-PE-Cy7 and Linage-PacBlue (CD45, Ter119 and CD31) (Biolegend). Viable cells were identified based on forward- and side- scatter profiles and by DAPI exclusion. Single cells were analyzed and sorted using FACS Aria II (BD Bioscience). For in vitro colony forming assay, 10.000 sorted epithelial cells were plated into 96-well plate previously coated with 2.5% growth factor reduced matrigel. If not otherwise specified, irradiated L1-Wnt3a expressing cells were used as feeder layer. Cells were grown into liquid media supplemented with Y-27632 dihydrochloride (Sigma-Aldrich), murine recombinant EGF (Prepotech), rhR-Spondin1 (R&D) and Nogging (R&D), as previously described^{10 (link)}.

Adorno M., di Robilant B.N., Sikandar S.S., Acosta V.H., Antony J., Heller C.H, & Clarke M.F. (2018). Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation. Scientific Reports, 8, 17506.

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Multicolor Flow Cytometry for Immune Cell Profiling

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PE anti-CD3, Pacific Blue anti-CD8α, biotin-conjugated anti-CD8β, PE Cy7 anti-CD4, APC Cy7 anti-CD62L, APC anti-CD103, PerCP Cy5.5 anti-TCRγδ, PE Cy7 anti-TCRβ, APC Cy7-B220, PerCP Cy5.5 anti-B220, FITC-EpCAM, PE Cy7 anti-CD11b, APC anti-F4/80, biotin-conjugated anti-IgA antibodies were purchased from BioLegend. Streptavidin-PE was purchased from BD Biosciences.

Ito K., Nakajima A., Fukushima Y., Suzuki K., Sakamoto K., Hamazaki Y., Ogasawara K., Minato N, & Hattori M. (2017). The potential role of Osteopontin in the maintenance of commensal bacteria homeostasis in the intestine. PLoS ONE, 12(3), e0173629.

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Colon Tissue Cell Profiling

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Tumor or normal colon tissue cells were digested into single cell as the method mentioned above, cells were subsequently counted, and the surface of the cells were stained for 30 min at room temperature using Fixable viability stain 510 (564406, 2 μg/mL, BD Biosciences, Franklin Lake, New Jersey, USA), FITC‐EPCAM (Cat. #324203, 9C4, 1 μg/mL, Biolegend, San Diego, CA, USA), PERCP‐CY5.5‐CD31(Cat. #303131, WM59, 1 μg/mL, Biolegend), APC‐hFAP (Cat. #FAB3715A‐025, R&D, Emeryville, CA, USA) were used for surface staining. Subsequent analysis was performed with FlowJo software (Tree Star Inc., San Carlos, CA, USA).

Chen S., Fan L., Lin Y., Qi Y., Xu C., Ge Q., Zhang Y., Wang Q., Jia D., Wang L., Si J, & Wang L. (2023). Bifidobacterium adolescentis orchestrates CD143+ cancer‐associated fibroblasts to suppress colorectal tumorigenesis by Wnt signaling‐regulated GAS1. Cancer Communications, 43(9), 1027-1047.

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