Cd31 pe

Manufactured by Miltenyi Biotec

Sourced in United States, Canada

CD31-PE is a monoclonal antibody conjugated with the fluorescent dye phycoerythrin (PE). It is used for the detection and analysis of CD31, also known as platelet endothelial cell adhesion molecule (PECAM-1), which is expressed on the surface of endothelial cells, platelets, and certain leukocyte subsets.

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Anti-CD31 PE (2 citations) Anti-CD31 PE-Cy7 (2 citations)

7 protocols using cd31 pe

Comprehensive Immunophenotyping of Cell Populations

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Cells were stained with the appropriate conjugated antibodies for 30 min at 4°C, washed in 0.5% bovine serum albumin/phosphate-buffered saline (PBS) 2 mmol/L EDTA solution, and analyzed using FACSCalibur (BD Biosciences). The following antibodies (anti-human) were utilized: PE CD31: Miltenyi Biotec #130-118-965; FITC CD34: Miltenyi Biotec #130-113-178; APC CD144: Miltenyi Biotec #130-126-010; APC CD73: Miltenyi Biotec #130-124-011; PE CD45 (clone HI30): BioLegend #304008; FITC CD3: Miltenyi Biotec #130-113-690; PE CD90: Miltenyi Biotec #130-117-388; BV421 CD56: BD Horizon #562751; AF647 CD158 (KIR): BD Pharmingen #567324; FITC Perforin: eBioscience #11-9994-42; KO CD45: Beckman Coulter #A96416; PE NKp46: BD Pharmingen #557991; PE-Cy7 CD159α (NKG2A): Beckman Coulter #B10246; PEDazzle CD16: BioLegend #302054; PerCP-Cy5 CD94: BioLegend #302054; FITC IFNγ: Sysmex #AE092283; PE TNFα: BD Pharmingen #554513; CF594 IL-8: BD Horizon #563531.

Galat Y., Du Y., Perepitchka M., Li X.N., Balyasnikova I.V., Tse W.T., Dambaeva S., Schneiderman S., Iannaccone P.M., Becher O., Graham D.K, & Galat V. (2023). In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies. Oncoimmunology, 12(1), 2240670.

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Multiparameter Flow Cytometry of MSCs and Macrophages

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For MSC cell-surface characterization, the following antibodies were used: APC-Vio770 CD29 (Miltenyi Biotec; clone, HMβ1-1), VioBright FITC CD44 (Miltenyi Biotec, USA; clone, REA665), PerCP Sca-1 (Biolegend, USA; clone, D7), PE CD31 (Miltenyi Biotec, USA; clone, 390), PE CD45 (Biolegend, USA; clone, 30-F11), and PE-Vio770 CD105 (Miltenyi Biotec, USA; clone, MJ7/19). For analysis of in vitro macrophages, we first blocked Fc receptors with CD16/32 FcγRIII/FcγRII (BD Bioscience, USA; clone, 2.4G2) and CD16.2 FcγRIV (Biolegend, USA; clone, 9E9) and then stained with PE CD45 (Biolegend, USA; clone, 30-F11), Alexa Fluor 647 CD206 (BioLegend, USA; clone, C068C2), and PE CD163 (ThermoFisher, USA; clone, TNKUPJ). A Novocyte 2060 (ACEA Biosciences, USA) was used to measure mean fluorescence intensity of GFP from transfected MSCs, MSC cell-surface markers, and macrophage markers. Data was analyzed in FlowJo software (BD Biosciences, USA).

Enam S.F., Kader S.R., Bodkin N., Lyon J.G., Calhoun M., Azrak C., Tiwari P.M., Vanover D., Wang H., Santangelo P.J, & Bellamkonda R.V. (2020). Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells. Journal of Neuroinflammation, 17, 197.

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Profiling Monocyte Surface Markers

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To study the surface cell markers on monocytes (CD14 +), PBMCs from the 10 patients used for single-cell analysis and 10 HDs were defrosted and washed once with PBS. After blocking for non-specific binding with Fc block (BD Pharmingen) for 5 min on ice, cells were incubated for 20 min on ice using staining buffer (PBS with 4% fetal bovine serum and 0.4% EDTA). Antibodies used included the following: CD14-FitC (Miltenyi Biotec), CD85-PEvio770 (Miltenyi Biotec), CD172a-APC (Miltenyi Biotec), CD97-PEvio770 (Miltenyi Biotec), CD31-PE (Miltenyi Biotec), CD366-PEvio615 (Miltenyi Biotec), CD62L-APC (Miltenyi Biotec), CD58-PE (Miltenyi Biotec), CD191-PEvio770 (Miltenyi Biotec), CD52-PEvio615 (Miltenyi Biotec), CD48-APC (Miltenyi Biotec). Cells were analyzed in a BD FACSCanto-II flow cytometer.

Godoy-Tena G., Barmada A., Morante-Palacios O., de la Calle-Fabregat C., Martins-Ferreira R., Ferreté-Bonastre A.G., Ciudad L., Ruiz-Sanmartín A., Martínez-Gallo M., Ferrer R., Ruiz-Rodriguez J.C., Rodríguez-Ubreva J., Vento-Tormo R, & Ballestar E. (2022). Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines. Genome Medicine, 14, 134.

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Multiparameter Flow Cytometry Analysis

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We used the following conjugated antibodies: CD38-fluorescein isothiocyanate, CD34-phycoerythrin (PE)-cyanin5 (Cy5), CD4-Pacific Blue, CD19-allophycocyanin (APC), CD3-Alexa Fluor 700, CD8-APC-H7, CCR7-Alexa Fluor 647, CD79a-APC from BD Biosciences (San Jose, CA); CD56-PE, CD45RA-PE-TexasRed, CD7-PE-Cy7, CD1a-PE, CD5-PE-Cy5 from Beckman Coulter (Brea, CA); CD31-PE from Miltenyi Biotech (Bergisch Gladbach, Germany) and CD27-PE-Cy7 from eBioscience (San Diego, CA). We obtained LIVE/DEAD aqua fluorescent dye from Molecular Probes (Invitrogen, Carlsbad, CA). The Fam-FLICA-FITC probe against active caspase-1 was used according to the manufacturers’ instructions (ImmunoChemistry, Bloomington, MN). Standard protocols were used for all types of staining. Data were acquired with an LSRII Flow Cytometer (BB Biosciences), and analyzed with FlowJo v7.6.5 (Treestar, Ashland, OR).

Menkova-Garnier I., Hocini H., Foucat E., Tisserand P., Bourdery L., Delaugerre C., Benne C., Lévy Y, & Lelièvre J.D. (2016). P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART. PLoS Pathogens, 12(4), e1005571.

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Multiparametric Flow Cytometry Immunophenotyping

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Adherent cells or EBs were dissociated to form a single-cell suspension by TrypLE (Life Technologies) treatment, and washed with FACS buffer (1% FBS and 1 mM EDTA in PBS). The dissociated cells were resuspended in FACS buffer, and labeled with fluorochrome-conjugated anti-human CD34-APC (clone# AC136, Miltenyi Biotec), CD31-PE (clone# AC114.5, Miltenyi Biotec), CD144-Alexa Fluor 700 (clone# 16B1, eBioscience), CD45-BV605 (clone# HI30, BioLegend), CD41-APC/Cy7 (clone# HIP8, BioLegened), CD235a-PE (clone# HIR2, eBioscience), CD43-BV421 (clone# 1G10, BD Biosciences), CD73-PE/Cy7 (clone# AD2, BioLegend), CD117-PE/Cy7 (clone# 104D2, BioLegend), CD127-FITC (clone# eBioRDR5, eBioscience), CD14-FITC (clone# 61D3, eBioscience), or KDR-PerCP (clone# HKDR-1, BioLegend). Flow cytometry was performed on LSR II analyzer (BD Biosciences). Data analysis was performed using FlowJo software or FCS Express software.

BAI H., LIU Y., XIE Y., HOYLE D.L., BRODSKY R.A., CHENG L., CHENG T, & WANG Z.Z. (2015). Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells. Journal of cellular physiology, 231(5), 1065-1076.

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Flow Cytometry Analysis of Endothelial Cells

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Cells were harvested and suspended in ice-cold PBS with 1% BSA and 2 mM EDTA. After incubation with FcR Blocking Reagent (Miltenyi Biotec), cells were stained by fluorescently conjugated antibodies and incubated for 10 min in the dark in the refrigerator (2 − 8 °C). Antibodies include CD31-PE, CD144-APC, CD146-PE CD248-APC, IgG-APC and IgG-PE from Miltenyi Biotec (antibody information and dilution used is listed in the supplementary table 1). DAPI staining was used for dead cell exclusion. The stained cells or GFP and mCherry-labeled cells were analyzed in a BD Fortessa analyzer. FACS sorting was performed using the BD FACSAria cell sorter. Data were analyzed using FlowJo v10.8 software (https://www.flowjo.com/). A representative gating strategy for sorting mCherry+ cells is included in the source data.

Muthukrishnan S.D., Kawaguchi R., Nair P., Prasad R., Qin Y., Johnson M., Wang Q., VanderVeer-Harris N., Pham A., Alvarado A.G., Condro M.C., Gao F., Gau R., Castro M.G., Lowenstein P.R., Deb A., Hinman J.D., Pajonk F., Burns T.C., Goldman S.A., Geschwind D.H, & Kornblum H.I. (2022). P300 promotes tumor recurrence by regulating radiation-induced conversion of glioma stem cells to vascular-like cells. Nature Communications, 13, 6202.

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Endothelial Marker Expression Analysis

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To verify the endothelial marker expression the iECs were analyzed via flow cytometry. The cells were harvested with StemPro Accutase (ThermoFisher Scientific), washed with ice-cold FACS buffer (PBS + 1% FBS + 2 mM EDTA), and incubated with conjugated antibodies CD31 PE, CD34 FITC, VE-Cadherin APC (Miltenyi Biotech) for 30 minutes at 4°C. Following this, the cells were washed with a 0.5% BSA/PBS solution. Data collection was performed via the FACSCalibur (BD Biosciences) and analyzed with the FlowJo software (version 10.5.3).

Perepitchka M., Galat Y., Beletsky I.P., Iannaccone P.M, & Galat V. (2020). Down syndrome iPSC model: endothelial perspective on tumor development. Oncotarget, 11(36), 3387-3404.

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