Cd45 pe cy5

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

CD45-PE-Cy5 is a fluorescently labeled antibody that targets the CD45 antigen, a common leukocyte antigen expressed on the surface of various white blood cells. It is designed for use in flow cytometry applications to identify and quantify CD45-positive cell populations.

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CD45-PE (59 citations) Anti-CD45 Pe-Cy5 (3 citations)

6 protocols using cd45 pe cy5

Flow Cytometry Analysis of Tumor Immune Cells

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Tumor tissue was digested in a 0.3% collagenase/0.1% hyaluronidase solution, pressed through a nylon mesh filter to obtain a single cell suspension and incubated in red cell lysis buffer (0.17 M Tris-HCl, 0.16 M NH₄Cl) for 3 min, spun down, and resuspended in FACS buffer (PBS + 1.5% FBS). Equal numbers of cells were stained with a viability dye and combinations of the following antibodies: CD8a-PECy7 (eBioscience, San Diego, CA, USA, 25-0081-82), F4/80-PECy7(eBioscience, 25-4801-82), CD206-FITC (Biolegend, 141704), Ly6G-APC (eBioscience, 17-5931-81), CD11b-PE (eBioscience, 12-0112-82), CD45-PE-Cy5 (eBioscience, 15-0451-83), IFN-γ-APC (eBioscience, 17-7311-82), and SIINFEKL pentamer-PE (ProImmune, Oxford, UK). For autophagy analysis using Cyto-ID staining of bone marrow derived macrophages, cells were first stained for surface markers (CD80-PE-Cy5 (eBioscience, 15-0801-81) and CD206-APC (eBioscience, 17-2061-82)) followed by staining with Cyto-ID for 30 min at 37 °C, per the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY, USA, ENZ-51031). Flow cytometric data were acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software version 9.2 (BD Biosciences, San Jose, CA, USA).

Alexander E.T., Fahey E., Phanstiel O I.V, & Gilmour S.K. (2023). Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice. Biomedicines, 11(10), 2703.

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Immunophenotyping of Mesenchymal Cells

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BM-MSC, fibroblasts or myofibroblasts were harvested using acutase, and incubated on ice for 1 h with directly conjugated antibodies including; SSEA-4-FITC (R&D Systems), CD44-PE (BD Bioscience), CD90-PE, CD105-APC, CD14-APC, CD45-PECy5 (eBioscience, Hatfield, UK), CD73-PE (BD Biosciences, Oxford, UK). Indirect staining was performed for GD2 (BD Biosciences), with a secondary goat anti-mouse-Alexa488 conjugated antibody at 1:200 for 40 min (Life Technologies). Cells were analysed using a FACScanto cytometer (Beckton Dickinson, Oxford, UK), and data analysed using FACS Diva (v6.2).

Chowdhury R., Webber J.P., Gurney M., Mason M.D., Tabi Z, & Clayton A. (2014). Cancer exosomes trigger mesenchymal stem cell differentiation into pro-angiogenic and pro-invasive myofibroblasts. Oncotarget, 6(2), 715-731.

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Multi-Parametric Flow Cytometry Sorting

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Fluorescence compensation was performed to correct any spectral overlap. Propidium iodide (PI) was added to samples at 1 mg/mL as a viability assay. Fluorescence minus one samples were run first to establish gating strategy for sorting. The following panels were used for sorting: 1) SSC-A versus FSC-A to capture all cells; 2) FSC-W versus FSC-H to capture singlets; 3) SSC-W versus SSC-H to capture singlets; 4) PI versus CD45-PECy5 (eBioscience, cat. no 15–0451-82) to capture all live (PI-), hematopoietic (CD45+) cells; 5) B220-PE (eBioscience, cat. no 12–0452-82) versus MAC1-BV421 (BD Horizon, cat. no 560456) to capture 3 populations: B-cells (B220+), macrophages (MAC1+), lymphocytic populations (B220– MAC1–). Representative FACs plots are shown in Figure 5A.

Moore A.L., desJardins-Park H.E., Duoto B.A., Mascharak S., Murphy M.P., Irizarry D.M., Foster D.S., Jones R.E., Barnes L.A., Marshall C.D., Ransom R.C., Wernig G, & Longaker M.T. (2020). Doxycycline Reduces Scar Thickness and Improves Collagen Architecture. Annals of surgery, 272(1), 183-193.

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Quantification of CD64 Expression in PBMC

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Blood samples were collected in tubes with EDTA anticoagulant. Whole blood was stained with anti-human CD45 phycoerythrin-cyanine5 (CD45-PE-Cy5; eBioscience, USA) and anti-human CD64 phycoerythrin (CD64-PE; Beckman Coulter, USA). After staining, red blood cells were lysed with Erythrocyte Lysis Buffer (BD Biosciences, USA) to obtain peripheral blood mononuclear cells (PBMC). PBMC stained with fluorescent antibodies were examined by flow cytometry (Cytomics™ FC 500, Beckman Coulter, USA). The data were analyzed by flow cytometry analysis software (FlowJo 7.6, LLC, USA). The scatter plot was drawn with side scatter (SSC) and CD45-PE-Cy5, and neutrophils and lymphocytes were gated to acquire their CD64 MFI. The CD64 index was determined by the ratio of the CD64 MFI of granulocytes to that of lymphocytes.

Tang Z., Qin D., Tao M., Lv K., Chen S., Zhu X., Li X., Chen T., Zhang M., Zhong M., Yang H., Xu Y, & Mao S. (2018). Examining the utility of the CD64 index compared with other conventional indices for early diagnosis of neonatal infection. Scientific Reports, 8, 9994.

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In Vivo Tumor Immune Cell Isolation

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For in vivo experiments, the tumors were weighed and digested at 37°C in 10 mL digestion solution (PBS supplemented with type I collagenase (200 U/mL), hyaluronidase and DNase I (100 µg/mL)) for 1 hour. Single cell suspensions were obtained by grinding the digested tissues and filtering through a 70 µm cell strainer (Becton and Dickinson, BD, USA). The immune cells were freshly isolated using density gradient centrifugation and stained with antibodies for 30 min at 4°C. The following monoclonal anti-mouse antibodies were used: CD45-PECy5.5 (eBioscience, USA), CD3-PECy7 (eBioscience, USA), CD4-APC (BioLegend, USA), CD8-APC (eBioscience, USA), F4/80-APC (Sungene, China), CD11b-PECy7 (eBioscience, USA) and MHC II-PE (eBioscience, USA). Flow cytometry was performed and the data were analyzed using FlowJo software (TreeStar, Ashland, Oregon, USA).

Xi Q., Zhang J., Yang G., Zhang L., Chen Y., Wang C., Zhang Z., Guo X., Zhao J., Xue Z., Li Y., Zhang Q., Da Y., Liu L., Yao Z, & Zhang R. (2020). Restoration of miR-340 controls pancreatic cancer cell CD47 expression to promote macrophage phagocytosis and enhance antitumor immunity. Journal for Immunotherapy of Cancer, 8(1), e000253.

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Tumor Immune Cell Isolation and Characterization

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The tumors were weighed, minced into small fragments, and digested at 37°C in 10 ml of digestion solution [PBS supplemented with type I Collagenase (200 U/ml), Hyaluronidase, and DNase I (100 μg/ml)] for 60 min. Single-cell suspensions were obtained by grinding the digested tissues and filtering them through a 70-μm cell strainer (BD Biosciences). The immune cells were isolated using Ficoll density gradient centrifugation. Freshly isolated immune cells were stained with antibodies for 30 min at 4°C. The following monoclonal anti-mouse antibodies were used: CD45-PECy5.5 (eBioscience), CD3-pecy7 (eBioscience), CD8-APC (eBioscience), NK1.1-APC (eBioscience), F4/80-APC (eBioscience), CD11b-pecy7 (eBioscience), CD11c-APC (eBioscience), and MHCII-PE (eBioscience). Flow cytometry was performed on a FACS Canto II flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (TreeStar, Ashland, OR).

Xi Q., Chen Y., Yang G.Z., Zhang J.Y., Zhang L.J., Guo X.D., Zhao J.Y., Xue Z.Y., Li Y, & Zhang R. (2020). miR-128 Regulates Tumor Cell CD47 Expression and Promotes Anti-tumor Immunity in Pancreatic Cancer. Frontiers in Immunology, 11, 890.

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