Anti mouse cd45.2 clone 104

Manufactured by BioLegend

Sourced in United States

Anti-mouse CD45.2 (clone 104) is a monoclonal antibody that recognizes the CD45.2 isoform of the mouse CD45 protein. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. The clone 104 antibody can be used to detect and analyze CD45.2-expressing cells.

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3 protocols using anti mouse cd45.2 clone 104

Tumor-Draining Lymph Node Analysis

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Tumor draining lymph nodes were harvested 7 days after the treatment and processed into single cell suspensions via mechanical crushing. After filtration (70 µm), cells (1 × 10⁶ cells) were aliquoted for staining. Cells were incubated with anti-mouse CD16/32 (clone 93; Biolegend; cat # 101301) to reduce non-specific staining. Cells were sequentially stained with iTag H-2Db HPV 16 E7 (RAHYNIVTF; cat # TB-5008-1), H-2Kb p15E (KSPWFTTL; cat # TB-M507-1) or H-2Kb Ova (SIINFEKL; cat # TB-5001-1) tetramers (MBL International, Woburn, MA, USA) for 30 min, followed by staining with anti-mouse CD45.2 (clone 104; cat # 109815) from Biolegend and anti-CD8 (clone KT15; cat # D271-A64) from MBL. Dead cells were excluded on analysis following sytox blue (Thermo Fisher Scientific; cat # S34857) staining. Isotype control antibodies and fluorescence-minus-one approaches were used to ensure staining specificity. All analyses were performed on a BD Fortessa analyzer running FACSDiva software and interpreted using FlowJo (vX10.0.7r2).

Okada R., Furusawa A., Vermeer D.W., Inagaki F., Wakiyama H., Kato T., Nagaya T., Choyke P.L., Spanos W.C., Allen C.T, & Kobayashi H. (2021). Near-infrared photoimmunotherapy targeting human-EGFR in a mouse tumor model simulating current and future clinical trials. EBioMedicine, 67, 103345.

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Multiparameter Flow Cytometry Analysis

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MOC cells were harvested and non-specific binding was blocked with anti-CD16/32 antibodies (Biolegend) prior to staining. Tissues were prepared into single-cell suspensions as previously described (14 (link)), followed by anti-CD16/32 antibody staining. Cell surface staining was performed using flourophore conjugated anti-mouse CD45.2 clone 104, CD3 clone 145-2C11, CD4 clone GK1.5, CD8 clone 53-6.7, NK1.1 clone PK136, CD31 clone 390, PD-L1 clone 10F.9G2, H2-K^b clone AF6-88.5, CD107a clone 1D4B, CD69 clone H1.2F3, PD-1 clone RMP1-30, CD11b clone M1/70, CD11c clone N418, CD80 clone 16-10A1, and CD86 clone GL-1 antibodies from Biolegend, 41BB clone 17B5 and OX40 clone OX-86 from eBioscience, and CD8 clone KT15 from MBL (Woburn, MA). H2Kb:KSPWFTTL (p15E_604–611) tetramer was purchased from MBL. Dead cells were excluded via 7AAD negativity. Isotype control antibodies and a “fluorescence minus one” method of antibody combination were used for specific staining validation. Data was acquired on a FACSCanto using FACSDiva software (BD Biosciences) and analyzed on FlowJo software vX10.0.7r2.

Moore E., Clavijo P.E., Davis R., Cash H., Van Waes C., Kim Y, & Allen C. (2016). Established T-cell inflamed tumors rejected after adaptive resistance was reversed by combination STING activation and PD-1–pathway blockade. Cancer immunology research, 4(12), 1061-1071.

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Single-Cell Tumor Immune Profiling

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Tumor tissues were processed into single-cell suspensions by mincing, as well as chemical (Murine Tumor Dissociation Kit, Miltenyi Biotec) and mechanical (gentleMACS Dissociator) dissociation, per manufacturer recommendations. Suspensions were filtered through a 100 μm filter and washed with 1% BSA in PBS prior to blocking nonspecific staining with anti-CD16/32 (BioLegend) antibody. Cell surface staining was performed using fluorophore-conjugated anti–mouse CD45.2 (clone 104), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD31 (clone 390), PDGFR (clone APA5), PD-L1 (clone 10F.9G2), H2-K^b (clone AF6-88.5), CD107a (clone 1D4B), PD-1 (clone RMP1-30), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone HK1.4), CTLA-4 (clone 9H10), CD39 (clone Duha59), CD11c (clone N418), F4/80 (clone BM8), and CD206 (clone C068C2) from BioLegend, and IL-12Rb2 (clone 305719) from R&D Systems. FoxP3⁺ Treg staining performed with the mouse Treg Staining Kit #1 (eBioscience) as per manufacturer’s protocol. Cell viability was assessed via staining with Sytox (Thermo Fisher Scientific) or Zombie (BioLegend) dyes. All analyses were performed on a BD Fortessa analyzer running FACSDiva software and interpreted using FlowJo V.X10.0.7r2.

Hong Y., Robbins Y., Yang X., Mydlarz W.K., Sowers A., Mitchell J.B., Gulley J.L., Schlom J., Gameiro S.R., Sievers C, & Allen C.T. (2022). Cure of syngeneic carcinomas with targeted IL-12 through obligate reprogramming of lymphoid and myeloid immunity. JCI Insight, 7(5), e157448.

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