Cd31 pe

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CD31-PE is a laboratory instrument used for the detection and quantification of CD31 (platelet endothelial cell adhesion molecule-1) in cell samples. CD31-PE utilizes a fluorescent dye, phycoerythrin (PE), to label the CD31 antigen, enabling its identification and analysis through flow cytometry or similar techniques.

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CD31-PE (clone 390; (4 citations) Anti-mouse CD31(PECAM-1)-PE (2 citations) CD31-PE-Cy7 (27 citations) PE-conjugated anti-mouse PeCAM1 (CD31) antibody (2 citations) Rat anti-mouse CD31-PE (3 citations) Anti-CD31-PE-Cyanine7 (WM-59, (2 citations) Anti-CD31-PE (2 citations) CD31 PE-Cy7 (clone 390, (2 citations) CD31 (PE. anti-mouse, (4 citations) Anti-CD31-PE-Cy7 (9 citations)

24 protocols using cd31 pe

Characterization of PPAR-stimulated iMSC-Derived Extracellular Vesicles

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Pan PPAR-iMSC-EVs were stained using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). For analyzing the effect of pan PPAR-iMSC-EVs on hepatocyte regeneration, the hepatocytes were stained with anti-human CD90 APC-Cy7 antibody (BioLegend, San Diego, CA, USA) after pan PPAR-iMSC-EVs treatment and analyzed using the Attune NxT flow cytometer (Thermo Fisher Scientific). To confirm whether pan PPAR agonist-stimulated iMSCs express the typical cell surface markers for MSCs, pan PPAR agonist-stimulated iMSCs were stained with CD73 APC, CD105 PE, CD45 FITC, CD31 PE, and CD34 APC (eBioscience, Waltham, MA, USA) and CD90 APC-Cy7 (BioLegend) antibodies. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific).

Kim J., Lee S.K., Jeong S.Y., Cho H.J., Park J., Kim T.M, & Kim S. (2021). Cargo proteins in extracellular vesicles: potential for novel therapeutics in non-alcoholic steatohepatitis. Journal of Nanobiotechnology, 19, 372.

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Single Cell Sorting and Analysis

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Single cell suspensions were analyzed and sorted using a BD FACSJazz (Becton Dickinson) sorter as previously described. (Frank et al., 2016 (link); Zacharias et al., 2018 (link); Zepp et al., 2017 ) Briefly, single cell suspensions were stained with CD45-PerCP-Cy5.5 (eBioscience), CD31-PE (eBioscience) and EpCAM-APC (eBioscience) each at 1:1000. Cells were sorted into cold FACS Buffer and kept on ice for downstream applications. Flow cytometry analysis was performed on recorded events using FlowJo 10.

Penkala I.J., Liberti D.C., Pankin J., Sivakumar A., Kremp M.M., Jayachandran S., Katzen J., Leach J.P., Windmueller R., Stolz K., Morley M.P., Babu A., Zhou S., Frank D.B, & Morrisey E.E. (2021). Age dependent alveolar epithelial plasticity orchestrates lung homeostasis and regeneration. Cell stem cell, 28(10), 1775-1789.e5.

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Fibroblast Isolation from Adult Mice

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Liver, heart, lung, kidney, tail, gonad, and ventral skin of adult mice and E16.5 embryos were dissected and finely minced. Fibroblasts were isolated using enzymatic digestion with 0.05% Trypsin/Ethylenediaminetetraacetic acid (EDTA)(Gibco) under agitation at 37°C for 30–40 min. Cells were spun and plated in 10 cm² dishes and cultured to semiconfluence in Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher) high glucose supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher), 1% sodium pyruvate (Thermo Fisher), 1% penicillin–streptomycin (10,000 U/ml) (Thermo Fisher), 1% GlutaMAX Supplement (Thermo Fisher) in a 5% CO₂ incubator at 37°C. Passage 0 cells were then Trypsinized using TrypLE (Thermo Fisher) and further processed for flow cytometry, labeled using CD90-AF647 (BioLegend), CD45-PeCy7, and CD31-Pe (eBioscience) in 2% FBS in Hank's balanced salt solution (HBSS) (Thermo Fisher) and sorted using Influx or Aria II Sorter (BD). The CD90+; CD45−; CD31− fraction was collected for mRNA isolation (Figure 1—figure supplement 1). Adult fibroblasts from ROSA^Cas9-EGFP and Col1a1-GFP were sorted using CD90-APC (BioLegend), CD45-APCCy7(BioLegend), and CD31-PeCy7 (BD) after 3 or 5 days, respectively.

Forte E., Ramialison M., Nim H.T., Mara M., Li J.Y., Cohn R., Daigle S.L., Boyd S., Stanley E.G., Elefanty A.G., Hinson J.T., Costa M.W., Rosenthal N.A, & Furtado M.B. (2022). Adult mouse fibroblasts retain organ-specific transcriptomic identity. eLife, 11, e71008.

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Phenotypic analysis of aNSCs/NPCs

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The sub-ventricular zone was isolated and dissociated as described above for “Single cell RNA-seq using the 10x Genomics Chromium single cell technology”. In some cases, mice were given a 1mg/mouse dose of 5-ethynyl-2’-deoxyuridine (EdU) via intraperitoneal injection 4 hours prior to euthanasia. Antibody staining was carried out in FACS buffer at the following dilutions: PROM1-Biotin (eBioscience Cat.#13–1331-80 [1:300]), EGF-AlexaFluor 647 (Life Technologies Cat. #E35351 [1:300]), CD24-PacBlue (eBioscience Cat.#48–0242-80 [1:400]), CD31-PE (eBioscience Cat.#12–0311-81 [1:50]), CD45-BV605 (Biolegend Cat.#103139 [1:100]), Strep-PECy7 (eBioscience Cat.#25–4517-82 [1:500]), O4-PE (Miltenyi Cat.#130–099-211 [1:50]), CD317-FITC (Biolegend Cat.#127002 Clone:927 [1:50]) for one hour at 4°C. Samples were washed with 5mL of FACS buffer. Secondary staining was performed using Strep-PECy7 (eBioscience Cat.#25–4517-82 [1:500]) in FACS buffer at 4°C. Samples were washed with 5mL of FACS buffer, and resuspended in media containing 1μg/mL propidium iodide (Biolegend). Fluorescent-minus-one controls were used to set positive gates in each experiment. Cell populations were defined as follows:
BST2-positive and BST2-negative aNSCs/NPCs were subjected to subsequent analyses including bulk RNA-sequencing, EdU cycle analysis, and STAT1 staining.

Dulken B.W., Buckley M.T., Negredo P.N., Saligrama N., Cayrol R., Leeman D.S., George B.M., Boutet S.C., Hebestreit K., Pluvinage J.V., Wyss-Coray T., Weissman I.L., Vogel H., Davis M.M, & Brunet A. (2019). Single cell analysis reveals T cell infiltration in old neurogenic niches. Nature, 571(7764), 205-210.

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Isolation of Murine Lung Cell Types

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Murine lung tissue was digested into a single-cell suspension as previously described using dispase (Corning catalog #354235), collagenase (Roche catalog #10103578001), and DNase (Roche catalog #10104159001). When isolating AT2 cells from Sftpc^CreERT2:R26R^TdTomato mice, the animals received 200μg/gm tamoxifen (Sigma-Aldrich catalog #T55648) in corn oil (Sigma-Aldrich catalog #C8267) via gavage 3 days prior to being euthanized. Tomato+ cells were isolated using flow cytometry (MoFlow Astrios with Summit v 6.3.0.16900 software). To isolate AT2 cells from mice we stained single-cell suspensions of murine lung tissue and gated based on the following criteria: positive for EpCAM-APC (BioLegend catalog #118213) and negative for CD31-PE (eBioscience catalog #12-0311-81), CD45-PE (eBioscience catalog #12-0451-81), podoplanin-PE (eBioscience catalog #12-5381-80), Sca1-PE (eBioscience catalog #12-5981-81), CD24-PE (BioLegend catalog #119307), and DAPI (BioLegend catalog #422801) as previously described^{81 (link)}. To isolate mesenchymal cells we sorted for cells that were positive for CD140a (BioLegend catalog #135907) and negative for and DAPI (BioLegend catalog #422801). Antibody concentrations are in Supplementary Table 2.

Paris A.J., Hayer K.E., Oved J.H., Avgousti D.C., Toulmin S.A., Zepp J.A., Zacharias W.J., Katzen J.B., Basil M.C., Kremp M.M., Slamowitz A.R., Jayachandran S., Sivakumar A., Dai N., Wang P., Frank D.B., Eisenlohr L.C., Cantu E I.I.I., Beers M.F., Weitzman M.D., Morrisey E.E, & Worthen G.S. (2020). STAT3-BDNF-TrkB signaling promotes alveolar epithelial regeneration after lung injury. Nature cell biology, 22(10), 1197-1210.

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Phenotyping of Mesenchymal Stem Cells

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In order to identify the mesenchymal stem cell phenotype, approximately 5 × 10⁵ PDLSCs were incubated in 5% bovine serum albumin (BSA)/phosphate buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, USA) 1 × at 4 °C in dark for 1 h with the following monoclonal antibodies: PAR₁-FITC (Abcam, Cambridge, UK), OCT4-FITC (Abcam, Cambridge, UK), SOX2-FITC (Abcam, Cambridge, UK), STRO-1-FITC (Abcam, Cambridge, UK), CD14-FITC (eBioscience, San Diego, USA), CD90-FITC (eBioscience, San Diego, USA), CD31-PE (eBioscience, San Diego, USA), CD-44-PE (eBioscience, San Diego, USA), CD34-FITC (Biolegend, San Diego, USA) and CD146-PE (Biolegend, San Diego, USA) for 30 min at 4 °C. Unstained control was used to set gates. A total of 10–50,000 events were recorded and data analyzed through FlowJo (Becton Dickinson, Oregon, USA).

Alves T., Gasparoni L.M., Balzarini D., Albuquerque-Souza E., de Oliveira V., Rovai E.S., da Silva J., Huamán-Mendoza A., Catalani L.H., Sipert C.R, & Holzhausen M. (2022). Osteogenesis in human periodontal ligament stem cell sheets is enhanced by the protease-activated receptor 1 (PAR1) in vivo. Scientific Reports, 12, 15637.

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Isolation and Characterization of Rat MSCs

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Bone-derived MSCs (MSCs) were isolated and harvested from adult male Sprague Dawley rats (SD, Jiesijie, Co., Shanghai, China) weighing 200–250 g as previously described [23 (link)]. The cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, NY) with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) and incubated at 37 °C with 5% CO₂. Identification was performed by flow cytometry (BD Biosciences, Mississauga, ON) following the instructions. Cells were incubated in 100 μl PBS with CD29-APC (1:100 dilution, BD Biosciences, Mississauga, ON), CD90-cy5.5 (1:100 dilution, BD Biosciences), CD31-PE (1:100 dilution, eBioscience, San Diego, CA), FITC-CD45 (1:100 dilution, eBioscience), and their isotype control antibodies for 20 min on ice. After three washes with PBS, the cells were suspended in 300 μl of PBS and analyzed in fluorescence-activated cell sorter (FACS) instrument (BD Biosciences).

Cheng Z., Wang L., Qu M., Liang H., Li W., Li Y., Deng L., Zhang Z, & Yang G.Y. (2018). Mesenchymal stem cells attenuate blood-brain barrier leakage after cerebral ischemia in mice. Journal of Neuroinflammation, 15, 135.

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Immunophenotyping of Embryonic AGM Cells

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Single cells obtained from E11 AGMs were dissociated by collagenase. Then antibody staining was performed for 30 mins at 4°C using antibodies specific to c-Kit-APC, CD31-PE, CD41-FITC, CD45-APC, CD34-PE and TER119-PE-CY7 (eBioscience). The cells were analyzed with the MoFlo XDP (Beckman Coulter).

Zhou W., He Q., Zhang C., He X., Cui Z., Liu F, & Li W. (2016). BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development. eLife, 5, e18108.

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Mesenchymal Stem Cell Differentiation

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The entire reagents and chemical materials utilized in this work were of analytical grade. Propidium iodide (PI), Ribonuclease A, 3 (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dimethyl sulfoxide(DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin Streptomycin solution, and trypsin were purchased from Gibco Invitrogen (Grand Island, NY). L-Glutamine, β-glycerophosphate (10 mM) (Sigma), Alizarin Red S (Sigma), and dexamethasone (Sigma). Sodium selenite (Sigma), DMEM medium (Bioidea, Iran), Oil Red (Sigma-Aldrich), Trizol reagent (Thermo Scientific), Syber Green Master Mix (Thermo Scientific), CD105-Percp-Cy5.5 (Biolegend), CD44-PE (eBioscience), CD45-FITC (BD Bioscience), and CD31-PE (eBioscience). Euthanasia chamber (Orchid Scientific).

Rahimi B., Panahi M., Lotfi H., Khalili M., Salehi A., Saraygord-Afshari N, & Alizadeh E. (2023). Sodium selenite preserves rBM-MSCs’ stemness, differentiation potential, and immunophenotype and protects them against oxidative stress via activation of the Nrf2 signaling pathway. BMC Complementary Medicine and Therapies, 23, 131.

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Immunofluorescent Imaging of Islet Cells

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Isolated islets stained with fluorescently labeled antibodies CD31 PE (eBioscience) and B220 FITC (Biolegend) or CD3 FITC (Biolegend) for 90 minutes on ice and then fixed with 2% paraformaldehyde. Islets were imaged at 810nm using an Olympus FV100MPE as described previously (83 (link)–85 (link)). 100 xy planes of 509 µm by 509 µm with a resolution of 0.994 µm/pixel and 1-um z-spacing were acquired. Image analysis was performed using Imaris (Bitplane) and MATLAB (Mathworks). Images were linearly unmixed, as previously described (83 (link), 84 (link)).

Whitesell J.C., Lindsay R.S., Olivas-Corral J.G., Yannacone S.F., Schoenbach M.H., Lucas E.D, & Friedman R.S. (2022). Islet Lymphocytes Maintain a Stable Regulatory Phenotype Under Homeostatic Conditions and Metabolic Stress. Frontiers in Immunology, 13, 814203.

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