Cd45.2 clone 104

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

CD45.2 (clone 104) is a mouse anti-mouse monoclonal antibody that recognizes the mouse CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of all nucleated hematopoietic cells. This antibody can be used to identify and distinguish different leukocyte populations.

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14 protocols using cd45.2 clone 104

Multiparameter Flow Cytometry Immunophenotyping

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CD45R/B220 (clone RA3-6B2, 1:100, eBioscience), CD3 (145-2c11, 1:100, eBioscience), CD45.2 (clone 104, 1:100, eBioscience), CD4 (clone GK1.5, 1:100, eBioscience), CD8 (clone 53-6.7, 1:100, eBioscience), CD19 (clone eBio1D3, 1:100, eBioscience), CD5 (clone 53-7.3, 1:100, eBioscience), CD21 (clone eBio4E3, 1:100, eBioscience), CD43 (clone eBioR2/60, 1:100, eBioscience), IgD (clone 11–26, 1:100, eBioscience), Gr1 (clone RB6-8C5, 1:100, eBioscience), IgM (clone 11f41, 1:100, eBioscience), GL-7 (clone GL-7, 1:100, eBioscience), Mac1 (clone M1/70, 1:100, eBioscience).

Souroullas G.P., Fedoriw Y., Staudt L.M, & Sharpless N.E. (2017). Lkb1 deletion in murine B lymphocytes promotes cell death and cancer. Experimental hematology, 51, 63-70.e1.

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Comprehensive T Cell Phenotyping Protocol

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Human cells were stained with fluorescence-conjugated antibodies to CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone RPA-T8), CD45RA (clone HI100), CD45RO (clone UCHL1), CD62L (clone DREG–56), CCR7 (clone 150503) (BD Biosciences), and CD7 (clone 124-1D1, eBioscience). T cell subsets were enriched by negative selection with a RosetteSep Human T Cell Enrichment Cocktail (StemCell Technologies) from peripheral blood and ovarian cancer tissues. Naïve T cells were obtained with CD45RO bead–negative selection (Miltenyi Biotec) and sorted and confirmed by FACS with 98% purity. Mouse single-cell suspensions were prepared from thymus, bone marrow, peripheral blood, lymph nodes, and spleen and labeled with fluorescence-conjugated antibodies to CD3 (clone 17A2), CD4 (clone RM4–5), CD8 (clone 53-6.7), CD19 (clone 1D3), CD44 (clone IM7), CD45.1 (clone A20), CD62L (MEL–14), CD90 (clone 53-2.1) (BD Biosciences), CD45.2 (clone 104), and CD45RB (clone C363.16A, eBioscience). Then, if necessary, the cells were sorted (BD FACSAria) or acquired on an LSR II flow cytometer (BD Biosciences), and data were analyzed with DIVA software (BD Biosciences).

Xia H., Wang W., Crespo J., Kryczek I., Li W., Wei S., Bian Z., Maj T., He M., Liu R.J., He Y., Rattan R., Munkarah A., Guan J.L, & Zou W. (2017). Suppression of FIP200 and autophagy by tumor-derived lactate promotes naïve T cell apoptosis and affects tumor immunity. Science immunology, 2(17), eaan4631.

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Multiparameter Flow Cytometry for Immune Profiling

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In polarization and in vivo experiments, cells were first incubated with a fixable viability dye (65-0865-14, eBioscience) at 1:1,000 dilution in FACS buffer (PBS containing 1% bovine calf serum) for 10 min at room temperature. Samples were blocked using 5% normal mouse serum in FACS buffer for 10 min at 4°C. Cells were stained with antibodies for 30 min at 4°C at 1:200 dilution in FACS buffer (all from eBioscience): CD3 (clone 17A2), CD4 (clone GK1.5), CD45.1 (clone A20), and CD45.2 (clone 104). Following staining, cells were washed and fixed with Foxp3/Transcription Factor Fixation/Permeabilization buffer (00-5521, eBioscience), then stained for intracellular proteins in 1× permeabilization buffer for 30 min at 4°C. The following antibodies were used at 1:100 dilution: IFN-γ (clone XMG1.2), IL-17A (clone eBio17B7), and Foxp3 (clone FJK-16s). Data were acquired using a BD FACSCanto flow cytometer and analyzed with FlowJo software.

Lange S.M., McKell M.C., Schmidt S.M., Hossfeld A.P., Chaturvedi V., Kinder J.M., McAlees J.W., Lewkowich I.P., Way S.S., Turner J, & Qualls J.E. (2017). l-Citrulline Metabolism in Mice Augments CD4+ T Cell Proliferation and Cytokine Production In Vitro, and Accumulation in the Mycobacteria-Infected Lung. Frontiers in Immunology, 8, 1561.

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Quantification of Antigen-Specific CD8+ T Cells

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Allophycocyanin-labelled dextramers specific for OVA H-2Kb (257-SIINFEKL-264) were purchased from Immudex (Copenhagen, Denmark). Samples for flow cytometry were stained with the appropriate antibody cocktails in ice-cold PBS supplemented with 2 mM EDTA and 1% FBS. Anti-mouse CD45 (clone 30F11), CD8α (clone 53-6.7), CD103 (clone 2E7), CD44 (clone IM7), CD45.1 (clone A20) and CD45.2 (clone 104) antibodies were obtained from eBioscience. Anti-mouse CD62L (clone MEL-14) and CD69 (clone H1.2F3) antibodies were obtained from BD Biosciences. Anti-mouse CD279 (PD-1, clone 29F.1A12) was obtained from Biolegend. Events were acquired using an LSRFortessa SORP (Becton Dickinson) flow cytometer or Spectral Cell Analyzer SP6800 (Sony) and data were analysed using FlowJo V10 software (Tree Star).

Enamorado M., Iborra S., Priego E., Cueto F.J., Quintana J.A., Martínez-Cano S., Mejías-Pérez E., Esteban M., Melero I., Hidalgo A, & Sancho D. (2017). Enhanced anti-tumour immunity requires the interplay between resident and circulating memory CD8+ T cells. Nature Communications, 8, 16073.

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Isolation and Analysis of Hepatic Immune Cells

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Fresh liver was perfused with 5 mL of saline through the portal vein at 100‐300 mg, then digested with Roswell Park Memorial Institute 1640 medium with deoxyribonuclease I and collagenase IV at 37^oC. After digestion, hepatocytes were pelleted and discarded by centrifugation at 50g for 5 minutes. Nonparenchymal cells were washed with Roswell Park Memorial Institute 1640 medium and pelleted by centrifugation at 350g for 15 minutes, then washed and blocked with 10% mouse serum for 30 minutes. Antibodies against cluster of differentiation 11b (CD11b; clone M1/70; Ebioscience, Hatfield, UK), Ly‐6C (clone HK1.4; Ebioscience), CD45.2 (clone 104; Ebioscience), F4/80 (dilution 1:50; clone BM8; Invitrogen), and Ly‐6G (clone 1A8; Biolegend, San Diego, CA) were added for 30 minutes in a dilution of 1:100 unless otherwise specified. Cell viability was assessed with Fixable Viability Dye eFluor780 (Ebioscience) according to the manufacturer's protocols. Cells were formalin‐fixed and analyzed by flow cytometry using a BD LSR Fortessa II (BD Bioscience, Oxford, UK).

Zou X., Ramachandran P., Kendall T.J., Pellicoro A., Dora E., Aucott R.L., Manwani K., Man T.Y., Chapman K.E., Henderson N.C., Forbes S.J., Webster S.P., Iredale J.P., Walker B.R, & Michailidou Z. (2018). 11Beta‐hydroxysteroid dehydrogenase‐1 deficiency or inhibition enhances hepatic myofibroblast activation in murine liver fibrosis. Hepatology (Baltimore, Md.), 67(6), 2167-2181.

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Murine Bone Marrow Transplantation Assay

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For murine BM transplantation experiments, 12-week-old C57BL/6-CD45.2 mice were conditioned by irradiation alone or followed by injection of fludarabine and BM cells from congenic C57BL/6-CD45.1 mice were transplanted by a single intravenous injection within 48 hours from conditioning. In one experimental setting mice received a sublethal irradiation (200 or 400 cGy) with the infusion of 0.5 × 10⁶ donor cells/mouse and in the other one an irradiation of 850 cGy with 0.05 × 10⁶ donor cells/mouse.
For engraftment evaluation, 50 µL of PB were collected 4 and 6 weeks after transplant in heparin by tail bleeding, lysed with ACK buffer and stained with antibodies against murine CD45.1 (clone A20, eBiosciences) and CD45.2 (clone 104, eBiosciences).

Pievani A., Michelozzi I.M., Rambaldi B., Granata V., Corsi A., Dazzi F., Biondi A, & Serafini M. (2018). Fludarabine as a cost-effective adjuvant to enhance engraftment of human normal and malignant hematopoiesis in immunodeficient mice. Scientific Reports, 8, 9125.

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Multiparametric Flow Cytometry and Histochemical Analysis of Murine AML

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For flow cytometry analyses, fluorescent antibodies against murine CD45.2 (clone 104, eBiosciences) and against human CD45 (clone HI30), CD33 (clone P67.6), CD34 (clone 8G12), CD19 (clone SJ25C1) (BD Biosciences), and CD45 (clone HI30, Invitrogen) were used. The analyses were performed on a FACS CantoII (BD Biosciences).
Humeri of fludarabine-treated SCID/beige mice transplanted with primary human AML blasts were fixed in 4% formaldehyde in phosphate buffer, decalcified in 10% EDTA (Sigma-Aldrich) and routinely processed for paraffin embedding. Serial 5 µm-thick sections were stained with Hematoxylin-Eosin and stained with anti-human CD33 (# NCL-L-CD33, clone PWS44, 1:100; Novocastra™) and Myeloperoxidase (#NCL-MYELO, clone 59A5, 1:100, Novocastra™) antisera.

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Isolation and Phenotyping of Immune Cells

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Single cell suspensions were made from spleen, thymus, lymph nodes, bone marrow and from lavage of peritoneal cavity 24hr after injection of 1.5ml thioglycolate. For analysis of myeloid cells from spleen, organs were incubated for 30 minutes with Liberase (Sigma-Aldrich) according to manufacturer’s protocol. Cells were blocked with 10% normal mouse serum. 7-AAD (Life Technologies) was used to exclude dead cells.
Abs for the following surface markers were used in this study: CD11c (clone HL3), CD8b (clone 53-5.8), CD19 (clone 1D3), CD90.1 (clone H1S51), and FcγRI clone X54-5/7.1 (all from Becton Dickinson). CD3ε (145-2c11), B220 (RA3-6B2), and CD45.2 (clone 104) (all from eBioscience). CD4 (clone RM4-4), F4-80 (clone BM8), Ly6C (HK1.4), and Ly6G (clone 1A8) (all from Biolegend), FcgRIIb (clone Ly17.2 produced in house); FcγRIII (clone 275003, from R&D) and FcγRIV (clone 012, from Sino Biological).

Fransen M.F., Benonisson H., van Maren W.W., Sow H.S., Breukel C., Linssen M.M., Claassens J.W., Brouwers C., van der Kaa J., Camps M., Kleinovink J.W., Vonk K.K., van Heiningen S., Klar N., van Beek L., van Harmelen V., Daxinger L., Nandakumar K.S., Holmdahl R., Coward C., Lin Q., Hirose S., Salvatori D., van Hall T., van Kooten C., Mastroeni P., Ossendorp F, & Verbeek J.S. (2018). A Restricted Role of FcγR in the Regulation of Adaptive Immunity. Journal of immunology (Baltimore, Md. : 1950), 200(8), 2615-2626.

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Antibody-Based Immune Cell Profiling

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Fluorescent-dye-labeled antibodies against CD45.1 (clone A20; BioLegend), CD45.2 (clone 104; eBioscience), CD4 (clone GK1.5; BioLegend), CD8 (clone 53.6.7; BioLegend), CD11b (clone M/170; eBioscience), TER119 (clone TER-119; BioLegend), B220 (clone 6D5; BioLegend), CD3 (clone 145-2C11; BioLegend), Gr1 (clone RB6-RC5; BioLegend), CD41 (clone MWReg30; BioLegend), CD48 (clone HM48-1; BioLegend), CD150 (clone TC15-12F12.2; BioLegend), c-Kit (clone 2B8; BioLegend), and Sca-I(clone D7; eBioscience) were purchased from eBioscience or BioLegend. Phospho-S6 (Ser235/236, clone D57.2.2E) Alexa Fluor 647 was purchased from CST. Blocking antibody for FcγR (clone 2.4G2) was obtained from Bio X Cell. Raptor (clone E6O3A) rabbit mAb and TSC1 (clone D43E2) rabbit mAb used in immunoblot were from CST.

Yin N., Jin G., Ma Y., Zhao H., Zhang G., Li M.O, & Peng M. (2022). SZT2 maintains hematopoietic stem cell homeostasis via nutrient-mediated mTORC1 regulation. The Journal of Clinical Investigation, 132(20), e146272.

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Comprehensive Immunophenotyping of Influenza Infection

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Lungs were harvested at the indicated time points and RNA was extracted by TRI-Reagent (Molecular Research Center). Viral loads were determined by RT-PCR using influenza virus specific primers as previously described (30 (link)). Cells were stained as previously described (31 (link)) using monoclonal antibodies against CD69 (clone H1.2F3), Thy1.1 (clone HIS51), Thy1.2 (clone 53-2.1), CD4 (clone L3T4), CD45.1 (clone A20), CD45.2 (clone 104), CD62L (clone MEL-14), CD44 (clone 1M7), CD127 (clone A7R34), KLRG1 (clone 2F1), IFN-γ (clone XMG1.2), TNF-α (clone MP6-XT22), and Granzyme-B (clone NGZB) (all from eBioscience, San Diego, CA), CD8α (clone 53-6.7, BD Biosciences, San Jose, CA), CD25 (clone PC61, BD Biosciences), and H-2^b MHC class I tetramers loaded with immunodominant influenza virus nuclear protein (NP) epitope NP_(366-374) or OVA_(257-264) peptide. Anti-CD16/32 (Fc Block, clone 2.4G2) (BD Biosciences) was used in all stains. For intracellular cytokine and granule staining, GolgiPlug (BD Biosciences), Cytofix/Cytoperm buffer (eBioscience) and Perm/Wash buffer (eBioscience) were used according to the manufacturer’s instructions. Samples were analyzed using a FACSAria flow cytometer (BD Biosciences). All data were analyzed using FlowJo software (Treestar).

Gracias D.T., Boesteanu A.C., Fraietta J.A., Hope J.L., Carey A.J., Mueller Y.M., Kawalekar O.U., Fike A.J., June C.H, & Katsikis P.D. (2016). Phosphoinositide 3-Kinases p110δ isoform regulates CD8+ T cell responses during acute viral and intracellular bacterial infections. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1186-1198.

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