Cd31 390

Manufactured by Thermo Fisher Scientific

CD31 (390) is a laboratory equipment product offered by Thermo Fisher Scientific. The core function of this product is to detect and quantify the expression of the CD31 protein, which is commonly used as a marker for endothelial cells. This equipment provides researchers with the necessary tools to study the role of CD31 in various biological processes, such as angiogenesis and cell-cell interactions.

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CD31-PE (clone 390; (4 citations) APC-anti-CD31 (clone 390, (4 citations) CD31 (clone 390; (4 citations) Anti-CD31 (390; (4 citations) CD31-FITC clone 390 (2 citations) CD31-biotin (clone 390, (2 citations) CD31 PE-Cy7 (clone 390, (2 citations) CD31-APC (390), (2 citations)

5 protocols using cd31 390

Multicolor Flow Cytometric Analysis

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For detection and analysis, the following antibodies/clones were used: FcɛR1a (MAR-1; BioLegend); CD117 (2B8; BioLegend); CD45 (30-F11; BioLegend), β7 integrin (M293; BD Biosciences); CD11b (M1/70; BioLegend); CD11c (N418; BioLegend); CD4 (GK1.5; BioLegend); CD8 (53–6.7; BioLegend); CD103 (2E7; BioLegend); EPCAM (G8.8; BioLegend); Thy1.2 (53–2.1; BioLegend); CD31 (390; eBioscience); TGF-β1 (Tw7-16B4; BioLegend); and mMCP-1 (RF6.1; eBioscience). Anti-mMCP-1 and isotype control were conjugated in parallel to Alexa Fluor 647 using a conjugation kit (Life Technologies). Intracellular staining was conducted using a BD Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer-supplied protocol. For flow cytometry, the cells were collected and stained for surface markers for 45 min before fixation. Intracellular mMCP-1 staining was conducted overnight at 4°C. All cell sorting was on a BD FACSAria Fusion cell sorter using BD FACSDiva software. For all flow cytometry not involving cell sorting, data were collected on a BD LSRII Fortessa or BD CANTO-II using BD FACSDiva software. All downstream data analysis was conducted in FlowJo.

Derakhshan T., Samuchiwal S.K., Hallen N., Bankova L.G., Boyce J.A., Barrett N.A., Austen K.F, & Dwyer D.F. (2020). Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation. The Journal of Experimental Medicine, 218(1), e20200321.

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Tumor Dissociation and Cell Characterization

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Tumors were minced using a razor and digested with 1 mg/ml collagenase A and collagenase D and 0.4 mg/ml DNase I in PBS at 37 °C for 2 h with rotation at 600 rpm in a thermomixer compact (Eppendorf). 10 mM EDTA was then added to stop the enzymatic reaction. The cell suspension was passed through a 70 μm filter and stained with live/dead fixable violet stain (Thermofisher Scientific). Cells were subsequently stained with the following fluorescently conjugated antibodies; CD45 (30-F11), Ly6G (1A8), F4/80 (BM8), CD11b (M/170), CD11c (N418), Thy1 (30-H12), Podoplanin (8.1.1.), PDGFRα (APA5; all from Biolegend) and CD31 (390; eBioscience) at 1:300 dilution. Flow cytometry was performed on LSR Fortessa (BD Biosciences) analyzers. Unstained and single-stained compensation beads (Invitrogen) were run alongside to serve as controls. Offline analysis was carried out on FlowJo (Treestar). For in vivo PAD4 inhibitor studies, tumors were separated for flow cytometric and immunofluorescent analysis. Some GSK484 treated tumors were too small for analysis and were therefore excluded (only tumor volumes were recorded).

Munir H., Jones J.O., Janowitz T., Hoffmann M., Euler M., Martins C.P., Welsh S.J, & Shields J.D. (2021). Stromal-driven and Amyloid β-dependent induction of neutrophil extracellular traps modulates tumor growth. Nature Communications, 12, 683.

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Multiparametric Flow Cytometry of Immune Cells

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Antibodies against the following antigens were used: CD11b (M1/70; eBioscience), CD49b (DX5; eBioscience), Siglec-F (E50-2440; BD), Ly6G/C (RB6-8C5; BD), FcεRI (Mar-1; BioLegend), CD45 (30-F11; BioLegend), CD34 (RAM34; eBioscience), ESAM-1 (1G8; BioLegend), CD31 (390; eBioscience), c-kit (2B7; BioLegend), CD106 (429; eBioscience), IL-4Rα (mIL4-M1; BD), and CD213a1 (13MOKA; eBioscience). Key cell populations were defined as follows: (a) eosinophils, FSC^loSSC^hiCD11b⁺Siglec-F⁺ or FSC^loSSC^hiSiglec-F⁺4get⁺; (b) basophils, FSC^loSSC^loCD49b⁺FcεRI⁺Gr-1⁻CD4⁻CD8⁻CD19⁻γδ⁻Siglec-F⁻ or FSC^loSSC^loBasoph8⁺CD49b⁺; (c) mast cells, 4get⁺ckit⁺ or FSC^hiSSC^hic-kit⁺CD11a⁻CD44⁺; (d) endothelial cells, CD45⁻CD34⁺ESAM-1⁺; and (e) PMNs/monocytes, Gr-1⁺CD11b⁺. Flow cytometry data acquisition was performed on an LSRII (BD), using FlowJo to analyze the data.

Cheng L.E., Sullivan B.M., Retana L.E., Allen C.D., Liang H.E, & Locksley R.M. (2015). IgE-activated basophils regulate eosinophil tissue entry by modulating endothelial function. The Journal of Experimental Medicine, 212(4), 513-524.

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Adipose Tissue Immune Cell Analysis

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Adipose tissue was digested in RPMI with 0.5% BSA and 1 mg/ml type II collagenase on a rocking platform for 25 min at 37 °C. The SVF was separated from adipocytes by centrifugation. The following antibodies were used for flow cytometry following Fcblock: CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8a (53-6.7), CD11c (N418), Sca-1 (Ly-6A/E) (D7), CD31 (390) (eBioscience), PDGFRα (RM0004-3G28) (Abcam), and CD64 (X54-5/7.1) (BD Pharmingen). For intracellular collagen staining, cells were incubated in Fc Block then stained with surface antibodies for 45 min at 4°C. Intracellular stains were performed using FOXP3 Fix/Perm buffer (BioLegend, San Diego, CA) followed by 15 min block with 0.5% goat serum, a 1hr incubation with 0.5μg of rabbit Collagen Type I antibody (Rockland, Limerick, PA), three washes, and 30 min incubation with 0.2μg of goat anti-rabbit AlexaFluor 647 antibody (Thermo Fisher Scientific-Life Technologies, Carlsbad, CA). Analysis was performed using a BD Biosciences FACSCanto II and FlowJo v.10 (Treestar).

Zamarron B.F., Porsche C.E., Luan D., Lucas H.R., Mergian T.A., Martinez-Santibanez G., Cho K.W., DelProposto J.L., Geletka L.M., Muir L.A., Singer K, & Lumeng C.N. (2020). Weight Regain in Formerly Obese Mice Hastens the Development of Hepatic Steatosis Due To Impaired Adipose Tissue Function. Obesity (Silver Spring, Md.), 28(6), 1086-1097.

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Multiparameter Flow Cytometry Analysis of Lung Cells

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BALF was collected by three tracheal instillations and aspirations of 1 mL PBS. Tissues were digested with collagenase D (1.5 U/mL) and dispase II (2.4 U/mL) (Roche) for 30 minutes. Samples were then incubated with anti-CD16/32 to block nonspecific antibody binding. Fluorescence-conjugated antibodies to CD45 (I3/2), CD11c (N418), CD3e (145-2C11), CD8 (53.67), CD4 (GK1.5), B220 (RA3-6B2), Ly6B (7/4) (Abcam), SiglecF (E50-2440) (eBiosciences), CD34 (RAM34) (eBiosciences), CD31 (390) (eBiosciences), PDGFRα (APA5) (eBiosciences), Sca1 (D7) (eBiosciences), and EpCAM (G8.8) (eBiosciences) were used. For EdU uptake experiments, mice were given 1 mg EdU daily by intraperiotenal injections; EdU detection was performed using the Click-IT assay kit (Life). Data was acquired on a BD LSRII and analyzed with FlowJo Software.
All antibodies were generated in-house (UBC AbLab) unless otherwise indicated.

Lo B.C., Gold M.J., Scheer S., Hughes M.R., Cait J., Debruin E., Chu F.S.F., Walker D.C., Soliman H., Rossi F.M., Blanchet M.R., Perona-Wright G., Zaph C, & McNagny K.M. (2017). Loss of Vascular CD34 Results in Increased Sensitivity to Lung Injury. American journal of respiratory cell and molecular biology, 57(6).

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