Anti cd45.2 v500

Manufactured by BD

Anti-CD45.2-V500 is a fluorochrome-conjugated antibody that binds to the CD45.2 antigen expressed on various mouse leukocyte populations. It can be used for the identification and enumeration of these cell types in flow cytometry applications.

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3 protocols using anti cd45.2 v500

Multiparametric Flow Cytometry Analysis

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Cells were stained with Zombie NIR™ Fixable Viability kit (BioLegend, 423106) according to the manufacturer’s protocol and blocked with anti-CD16 mAbs. For surface markers, subsequent antibodies were used: anti-CD45.2-V500 (104, BD Bioscience), anti-CD11b-FITC (M1/70, eBioscience), anti-Ly6C-PerCp-Cy7 (AL27, BD Bioscience), anti-Ly6G-APC (1A8, BioLegend), anti-CD3-V450 (17A2, eBioscience), anti-IL4R-PE (552509, BD Bioscience). For intracellular staining, after the cells were fixed and permeabilized with Cytofix/Cytoperm (554722, BD Bioscience), the following antibodies were applied: anti-IDO-eF660 (Mido-48, eBioscience) and anti-Arg1-PE (IC5868P, R&D). In order to analyze the Treg population, the Mouse Phenotyping Kit (560767, BD Bioscience) was used. Cells, resuspended in FACS flow buffer, were analyzed on FACSCanto II using Diva software. The cytokine concentration was measured in mouse serum, separated from the blood collected from the cheek vein. Serum was stained with BD™ Mouse Inflammation Kit, Cytometric Bead Array (552364, BD Bioscience).

Wachowska M., Stachura J., Tonecka K., Fidyt K., Braniewska A., Sas Z., Kotula I., Rygiel T.P., Boon L., Golab J, & Muchowicz A. (2020). Inhibition of IDO leads to IL-6-dependent systemic inflammation in mice when combined with photodynamic therapy. Cancer Immunology, Immunotherapy, 69(6), 1101-1112.

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Aim2 Knockout Immune Cell Analysis

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Spleen, thymus and inguinal lymph nodes were isolated from naive Aim2^+/+, Aim2^+/− and Aim2^−/− animals; tissue was gently dissociated and passed through a 70-µm cell strainer to generate single cell suspension; and red blood cells were lysed using ACK buffer. Cells were counted and stained using the following antibodies: anti-CD11b-FITC (cat. no. 11-0112, used at 1:200), anti-CD4-PerCpCy5.5 (cat. no. 45-0042, used at 1:80), anti-CD8-eFluor 450 (cat. no. 48-0083, used at 1:80), anti-CD11c-APC (cat. no. 17-0114, used at 1:80), anti-TCRβ-Alexa 700 (cat. no. 47-5961, used at 1:80), anti-F480-PE (cat. no. 12-4801, used at 1:80), anti-NK1.1-PECy7 (cat. no. 25-5941, used at 1:80) (eBioscience) and anti-B220-APC Cy7 (cat. no. 565371, used at 1:200) (BD Biosciences). Peripheral blood lymphocytes isolated from radiation bone marrow chimeric mice were stained with anti-CD45.1-PE (BD Biosciences, cat. no. 553776, used at 1:1,000) and anti-CD45.2-V500 (BD Biosciences, cat. no. 562130, used at 1:1,000). Flow cytometry was performed using an LSRII (BD Biosciences), and the resulting data were analyzed using FlowJo (TreeStar).

Wilson J.E., Petrucelli A.S., Chen L., Koblansky A.A., Truax A.D., Oyama Y., Rogers A.B., Brickey W.J., Wang Y., Schneider M., Mühlbauer M., Chou W.C., Barker B.R., Jobin C., Allbritton N.L., Ramsden D.A., Davis B.K, & Ting J.P. (2015). Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis by interfering with DNA-PK–dependent Akt activation. Nature medicine, 21(8), 906-913.

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Multicolor Flow Cytometry and Molecular Analyses of Immune Cells

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For flow cytometric analysis, anti-CD45.2-V500(562129, BD bioscience, 1:100), anti-CD45.2-APC(109814, BioLegend, 1:100), anti-CD4-PerCP/Cy5.5(100433, BioLegend, 1:100), anti-CD8a-PerCP/Cy5.5(100733, BioLegend, 1:100), anti-CD11b-PerCP/Cy5.5(45-0112-80, eBioscience, 1:100), anti-Ly6G-PerCP/Cy5.5(127615, BioLegend, 1:100), anti-B220-PerCP/Cy5.5(103235, BioLegend, 1:100), anti-CD11b-BV421(101251, BioLegend, 1:100), anti-CD64-APC(139306, BioLegend, 1:100), anti-F4/80-PE(123110, BioLegend, 1:100), anti-Ly6c-PE-Cy7(128018, BioLegend, 1:100) were used. For western blotting, anti-Cx40(AB1726, Merck Millipore, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-Cx45(AB1745, Merck Millipore, 1:2000), anti- α -tubulin(T6199, Sigma, 1:1000), anti-rabbit IgG-HRP(7074, Cell Signaling Technology, 1:5000), anti-mouse IgG-HRP(7076, Cell Signaling Technology, 1:5000) were used. For immunohistochemistry, anti-F4/80(MCA497G, Serotec, 1:500), anti-Ly6G(127601, BioLegend, 1:500), anti-B220 (103201, BioLegend 1:500), anti-CD3(GTX42110, GeneTex, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-N-cadherin(33-3900, ThermoFisher, 1:500) were used. The second antibodies used were Alexa-Fluor-488-conjugated anti-mouse-IgG(A-11001, ThermoFisher, 1:2000) and Alexa-Fluor-635-conjugated anti-rabbit-IgG(A-31576, ThermoFisher, 1:2000).

Sugita J., Fujiu K., Nakayama Y., Matsubara T., Matsuda J., Oshima T., Liu Y., Maru Y., Hasumi E., Kojima T., Seno H., Asano K., Ishijima A., Tomii N., Yamazaki M., Kudo F., Sakuma I., Nagai R., Manabe I, & Komuro I. (2021). Cardiac macrophages prevent sudden death during heart stress. Nature Communications, 12, 1910.

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