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Anti cd31 pe cy7

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Anti-CD31 PE-Cy7 is a fluorescently conjugated antibody that binds to the CD31 protein, also known as PECAM-1. CD31 is a cell surface marker expressed on endothelial cells, platelets, and certain leukocyte subsets. This antibody can be used to identify and quantify cells expressing CD31 in flow cytometry applications.

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Lab products found in correlation

Propidium iodide, by Merck Group (3 mentions) Fitc brdu flow kit, by BD (2 mentions) Anti cd 140a pdgfra, by Thermo Fisher Scientific (2 mentions) Brdu antibody, by BD (2 mentions) Anti cd45 pe cf594, by BD (2 mentions) Attune nxt software version 2, by Thermo Fisher Scientific (2 mentions) Attune nxt acoustic focusing flow cytometer, by Thermo Fisher Scientific (2 mentions) Dnase solution, by Worthington (2 mentions) Anti cd13 fitc, by BD (2 mentions) Anti cd45 pe cy7, by BD (2 mentions) Facsaria 2, by BD (2 mentions) Fortessa x20 flow cytometer, by BD (1 mentions) Anti ter119 pe cy7, by BD (1 mentions) Anti cd49f apc, by BioLegend (1 mentions) Anti sca 1 pe, by BD (1 mentions) Liberase, by Merck Group (1 mentions) Anti cd45 apc, by BD (1 mentions) Micro kit, by Qiagen (1 mentions) Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions)

11 protocols using anti cd31 pe cy7

1

Isolation and Analysis of Adipose-Derived Stromal Vascular Cells

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Fat depots were collected into 5mL of 1.0 mg/ml collagenase in ADB (w/o glucose and adenosine), minced with scissors and incubated in 37° water bath with shaking for 75 min, shook vigorously by hand and passed through 70um filter into 5mL of KRP buffer. Samples were spun at 300 g for 5min and processed for BRDU labeling using BD FITC BrdU Flow Kit (BD 559619). Briefly, The SVC pellet was resuspended in 200ul staining buffer and incubated with Anti-CD31 PE-Cy7 (BD Biosciences #561410), anti-CD45 PE-CF594 (BD Biosciences #562420) and Anti-CD-140a/PDGFRA, FITC (eBioscience, #11-1401-82) at RT (in dark) for 45min. Then, the samples were washed with 1ml of BD Perm/Wash buffer, spun at 300 g and the pellet resuspended and incubated for 30 min in 100ul in BD Cytofix/Cytoperm buffer. After one additional wash with BD Perm/Wash buffer, cells were resuspended and incubated in 100ul diluted DNase (30ul DNase + 70ul dPBS) at 37°C for 1hr. Then, the samples received BD BRDU antibody (BD 559619) and were incubated at 4°C overnight. Next, the cells were washed with 1ml BD Perm/Wash buffer, spun, resuspended with 250ul of staining buffer, passed through 70um filter and analyzed by flow cytometry, together with unstained and single stained controls.
Holland J., Sorrell J., Yates E., Smith K., Arbabi S., Arnold M., Rivir M., Morano R., Chen J., Zhang X., Dimarchi R., Woods S.C., Sanchez-Gurmaches J., Wohleb E, & Perez-Tilve D. (2019). A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake. Cell reports, 27(8), 2399-2410.e6.
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2

Isolation and Characterization of Cerebrovascular Cells

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Fresh cerebrovascular tissue was isolated from the brains of r-sham and r-mTBI mice at 6 months post-last injury and processed as previously described (Crouch and Doetsch, 2018 (link)). Briefly, the cerebrovascular pellet (obtained as described above) was resuspended in a collagenase/dispase solution (1 mg/ml) and incubated for 1 h at 37 °C with gentle agitation. Following enzymatic digestion, the tissue was pelleted by centrifugation at 6000 rpm for 3 min and resuspended in a DNAse solution (1 mg/ml, Worthington Biochemical) and subjected to further mechanical digestion via trituration with a pipette to achieve a single cell suspension. The tissue was centrifuged at 6000 rpm for 3 min, the supernatant discarded, and the resulting pellet was resuspended and stained with antibodies against the mural cell-specific N-aminopeptidase (CD13) using anti-CD13-FITC at 1:200 (BD Biosciences), and the brain endothelial cell marker (CD31) using anti-CD31-PE-CY7 at 1:500 (BD Biosciences). For live/dead cell discrimination, the viability dye propidium iodide (Sigma Aldrich) was added to the antibody cocktail. Cells were stained on ice for 30 min in the dark and resuspended in 1% BSA in HBSS. Data were acquired and analyzed using the Attune® NxT Acoustic Focusing Flow Cytometer and Attune® NxT software version 2.7 (Thermo Fisher Scientific, Waltham, MA, USA).
Ojo J., Eisenbaum M., Shackleton B., Lynch C., Joshi U., Saltiel N., Pearson A., Ringland C., Paris D., Mouzon B., Mullan M., Crawford F, & Bachmeier C. (2020). Mural cell dysfunction leads to altered cerebrovascular tau uptake following repetitive head trauma. Neurobiology of disease, 150, 105237.
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3

Isolation and Characterization of Mouse Mammary Epithelial Subpopulations

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Primary mouse mammary gland cells were used as controls for flow cytometry and were isolated using mechanical and enzymatic disaggregation as described previously [37 (link)]. The epithelial subpopulations were isolated from the disaggregated samples by flow cytometry as detailed previously [18 (link),19 (link),38 (link)]. Hematopoietic lineage cells were stained with anti-CD45-PE-Cy7, anti-TER119-PE-Cy7 and anti-CD31-PE-Cy7 (BD Biosciences, Franklin Lakes, NJ, USA). Cells were simultaneously stained with mammary-specific lineage markers, anti-Epcam BV421(BD Biosciences), anti-CD49f-APC (Biolegend, San Diego, CA, USA), anti-Sca-1-PE (BD Biosciences), anti-CD49b-FITC (Biolegend, San Diego, CA, USA) and propidium iodide (Sigma). Cells were analysed on a Fortessa-X20 flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Linear density contour plots were used to describe flow cytometry gates. Fluorescence-minus-one control gates defined marker-negative populations.
Lambouras M., Roelofs C., Pereira M., Gruber E., Vieusseux J.L., Lanteri P., Johnstone C.N., Muntz F., O’Toole S., Ooms L.M., Mitchell C.A., Anderson R.L, & Britt K.L. (2023). Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models. International Journal of Molecular Sciences, 24(6), 5666.
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4

Isolation and Characterization of Tumor Cells

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Tumors dissected from inguinal mammary glands free of the lymph node were minced, digested in Liberase (Sigma) and DNAse 1 reduced to single cell suspensions as described (31 (link)). Flow cytometry was performed using anti-CD3-PE (Clone 17A2), anti-B220-APC (Clone RA3-6B2), anti-mouse CD45-PE-Cy7 (Clone 30-F11), anti-F4/80-APC-eFluor 780 (Clone BM8), CD45 microbeads, anti-mouse CD31-APC (Clone 390), and anti-mouse CD105-PE (Clone MJ7/18). Macrophages were also labeled with fluorescent dextran via phagocytic uptake as previously described (32 (link)). Flow cytometric sorting of Endothelial cell with FACS Aria II (BD) was performed using anti CD45-APC (clone 30-F11) and anti CD31-PE-Cy7 (clone 390). Cells were collected in 1.5 ml tubes, centrifuged 10 min at 450 rcf and the pellet resuspended in cell lysis buffer. RNA extraction was performed using the Qiagen Micro Kit and retrotrascription performed with Superscript Vilo cDNA synthesis kit (Invitrogen).
Yeo E.J., Cassetta L., Qian B.Z., Lewkowich I., Li J.F., Stefater JA I.I.I., Smith A.N., Wiechmann L.S., Wang Y., Pollard J.W, & Lang R.A. (2014). Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer. Cancer research, 74(11), 2962-2973.
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5

Isolation and Analysis of Adipose-Derived Stromal Vascular Cells

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Fat depots were collected into 5mL of 1.0 mg/ml collagenase in ADB (w/o glucose and adenosine), minced with scissors and incubated in 37° water bath with shaking for 75 min, shook vigorously by hand and passed through 70um filter into 5mL of KRP buffer. Samples were spun at 300 g for 5min and processed for BRDU labeling using BD FITC BrdU Flow Kit (BD 559619). Briefly, The SVC pellet was resuspended in 200ul staining buffer and incubated with Anti-CD31 PE-Cy7 (BD Biosciences #561410), anti-CD45 PE-CF594 (BD Biosciences #562420) and Anti-CD-140a/PDGFRA, FITC (eBioscience, #11-1401-82) at RT (in dark) for 45min. Then, the samples were washed with 1ml of BD Perm/Wash buffer, spun at 300 g and the pellet resuspended and incubated for 30 min in 100ul in BD Cytofix/Cytoperm buffer. After one additional wash with BD Perm/Wash buffer, cells were resuspended and incubated in 100ul diluted DNase (30ul DNase + 70ul dPBS) at 37°C for 1hr. Then, the samples received BD BRDU antibody (BD 559619) and were incubated at 4°C overnight. Next, the cells were washed with 1ml BD Perm/Wash buffer, spun, resuspended with 250ul of staining buffer, passed through 70um filter and analyzed by flow cytometry, together with unstained and single stained controls.
Holland J., Sorrell J., Yates E., Smith K., Arbabi S., Arnold M., Rivir M., Morano R., Chen J., Zhang X., Dimarchi R., Woods S.C., Sanchez-Gurmaches J., Wohleb E, & Perez-Tilve D. (2019). A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake. Cell reports, 27(8), 2399-2410.e6.
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6

Identification of CA9+ Tumor Cells

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Cells were suspended in Hank’s balanced salt solution with 2 % FBS, blocked with 20 μg/ml mouse IgG on ice for 10 min, then incubated on ice with anti-CD31-PECy7 (1:100; BD Biosciences), anti-CD45-PECy7 (1:100; BD Biosciences) and anti-CA9-PE (Clone 303123, 1:10; R&D Biosystems) for 30 min, washed, and resuspended in Hank’s + 2 % FBS with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). Viable (i.e. DAPI-negative) CD45/CD31-negative cells were sorted into CA9+ and CA9− populations using a BD FACSAriaII cell sorter.
Lobo N.C., Gedye C., Apostoli A.J., Brown K.R., Paterson J., Stickle N., Robinette M., Fleshner N., Hamilton R.J., Kulkarni G., Zlotta A., Evans A., Finelli A., Moffat J., Jewett M.A, & Ailles L. (2016). Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine. BMC Cancer, 16, 485.
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7

Isolation and Characterization of Cerebrovascular Cells

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Fresh cerebrovascular tissue was isolated from the brains of r-sham and r-mTBI mice at 6 months post-last injury and processed as previously described (Crouch and Doetsch, 2018 (link)). Briefly, the cerebrovascular pellet (obtained as described above) was resuspended in a collagenase/dispase solution (1 mg/ml) and incubated for 1 h at 37 °C with gentle agitation. Following enzymatic digestion, the tissue was pelleted by centrifugation at 6000 rpm for 3 min and resuspended in a DNAse solution (1 mg/ml, Worthington Biochemical) and subjected to further mechanical digestion via trituration with a pipette to achieve a single cell suspension. The tissue was centrifuged at 6000 rpm for 3 min, the supernatant discarded, and the resulting pellet was resuspended and stained with antibodies against the mural cell-specific N-aminopeptidase (CD13) using anti-CD13-FITC at 1:200 (BD Biosciences), and the brain endothelial cell marker (CD31) using anti-CD31-PE-CY7 at 1:500 (BD Biosciences). For live/dead cell discrimination, the viability dye propidium iodide (Sigma Aldrich) was added to the antibody cocktail. Cells were stained on ice for 30 min in the dark and resuspended in 1% BSA in HBSS. Data were acquired and analyzed using the Attune® NxT Acoustic Focusing Flow Cytometer and Attune® NxT software version 2.7 (Thermo Fisher Scientific, Waltham, MA, USA).
Ojo J., Eisenbaum M., Shackleton B., Lynch C., Joshi U., Saltiel N., Pearson A., Ringland C., Paris D., Mouzon B., Mullan M., Crawford F, & Bachmeier C. (2020). Mural cell dysfunction leads to altered cerebrovascular tau uptake following repetitive head trauma. Neurobiology of disease, 150, 105237.
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8

Isolation of Murine Lung Endothelial Cells

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C57BL/6 mice were intravenously injected with 3 × 105 MC-38GFP cells. After 12 hours, lungs were perfused with PBS and digested with collagenase A and collagenase D (each 2 mg/ml, Roche) for 1 hour. Cells were filtered through a 70-μm cell strainer (BD), and the single cell suspension was stained with anti–CD45-Pacific Blue (Biolegend), anti–CD11b-APC-Cy7, anti–CD31-PE-Cy7, and anti–Ly6C-FITC (all BD). Endothelial cells (CD31+) were sorted.
Roblek M., Strutzmann E., Zankl C., Adage T., Heikenwalder M., Atlic A., Weis R., Kungl A, & Borsig L. (2016). Targeting of CCL2-CCR2-Glycosaminoglycan Axis Using a CCL2 Decoy Protein Attenuates Metastasis through Inhibition of Tumor Cell Seeding. Neoplasia (New York, N.Y.), 18(1), 49-59.
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9

Isolation of Tumor Cells and CAFs

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Tumors were collected and dissociated using 0.1% Collagenase type I (Thermo Fisher Scientific) and 0.2% Dispase type II (Sigma-Aldrich), filtered using a 70 mm cell strainer and centrifuged for 5 minutes at 1,500 RPM. Red blood cell lysis was performed by incubating the cell pellet for 3 minutes in 1 mL of Hybri-Max buffer (Sigma-Aldrich). Counted cells were resuspended in PBS containing 0.5% BSA and 2 mmol/L EDTA (FACS buffer) in a concentration of 10 6 cells/100 mL and incubated for 15 minutes with 1 mL of mouse BD Fc Block (BD Biosciences) per 1 million cells. Cells were stained with anti-Epcam-VioBlue (Miltenyi Biotec), anti-Pdgfra-APC (Thermo Fisher Scientific), anti-Cd31-PE-Cy7 (BD Biosciences) and Fixable viability dye eFluor780 (Thermo Fisher Scientific) for 20 minutes on ice, washed and resuspended in FACS buffer. Epcam þ tumor cells or Pdgfra þ CAFs were collected using a FACS Fusion (BD Biosciences). Data were analyzed using FlowJo v10 software (FlowJo LLC).
Mosa M.H., Michels B.E., Menche C., Nicolas A.M., Darvishi T., Greten F.R, & Farin H.F. (2020). A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer. Cancer research, 80(24).
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10

Quantifying Bone Marrow Chimerism

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To evaluate BM chimerism, BM mononuclear cells were isolated from donors, recipients and chimeras using Ficoll density gradient centrifugation, and stained with PE/Cy7-anti-CD31 (BD Biosciences, San Jose, CA) plus Alexa Fluor 488-anti-GFP (Life Technologies, Grand Island, NY) antibodies. Fluorescence minus one controls were stained in parallel in all analyses. Cells were analyzed using FACS to determine percentage of GFP+ EPCs in BM mononuclear cell population. Degree of donor chimerism was determined by comparing percentage of GFP+ EPCs between each chimera and its respective donor.
Mao S.Z., Ye X., Liu G., Song D, & Liu S.F. (2014). An Obligatory Role of NF-κB in Mediating Bone Marrow Derived Endothelial Progenitor Cell Recruitment and Proliferation Following Endotoxemic Multiple Organ Injury in Mice. PLoS ONE, 9(10), e111087.
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