Anti mouse gr 1

Manufactured by BioLegend

Anti-mouse Gr-1 is a monoclonal antibody that specifically binds to the Ly-6G and Ly-6C antigens, which are expressed on the surface of mouse granulocytes, including neutrophils, eosinophils, and a subpopulation of monocytes.

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6 protocols using anti mouse gr 1

Decitabine Effects on Leukemic Cells

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Peripheral blood was obtained from leukemic mice treated with vehicle or decitabine. After removing red blood cells using RBC lysis buffer, expression of GFP and tRFP were analyzed by a FACS Verse (BD Biosciences, San Jose, CA, USA). The sgTrp53-transduced and Trp53-deficient MLL-AF9 cells were treated with 50 nM decitabine or DMSO for 24 hours and were stained by fluoro-conjugated antibodies [anti-mouse CD117 (BioLegend, catalog #105808, clone 2B8, 1:400), and anti-mouse Gr-1 (BioLegend, catalog #108411, clone RB6-8C5, 1:400)] for 30 min at 4 °C. After staining, cells were washed with cold PBS several times, and were resuspended with PBS containing 2% FBS. Cells were analyzed on a FACS Verse (BD).

Tamura M., Yonezawa T., Liu X., Asada S., Hayashi Y., Fukuyama T., Tanaka Y., Kitamura T, & Goyama S. (2019). Opposing effects of acute versus chronic inhibition of p53 on decitabine’s efficacy in myeloid neoplasms. Scientific Reports, 9, 8171.

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Isolation and Characterization of Lung Cells

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IR-61 (2 mg kg⁻¹) was injected i.v. into mice. 24 h later, the lungs were minced and chopped to approximately 10 mm pieces. Then the tissues were digested in DMEM medium containing 2 mg mL⁻¹ of type IV collagenase (#C5138, Sigma), 0.125 mg mL⁻¹ DNase I (#C5025, Sigma) for 30 min at 37 °C and subsequently the resulting suspension passed through a 70 μm cell strainer. After red cell lysis, the cells were blocked with 1% of rat anti mouse CD16/CD32 (#553141, BD Biosciences, RRID: AB_394656) and then stain with anti-mouse CD3 (#100218, Biolegend, RRID: AB_1595492), anti-mouse CD11b (#101205, Biolegend, RRID: AB_312788), anti-mouse Gr1 (#108433, Biolegend, RRID:AB_10900232), anti-mouse CD19 (#115520, Biolegend, RRID: AB_313655), anti-mouse F4/80 (#123116, Biolegend, RRID: AB_893481), and anti-mouse CD11c (#117308, Biolegend, RRID: AB_313777) for 30 min at 4 °C. The acquisition was performed on a FACSCanto flow cytometer with FACSDiva software, and subsequent analysis was performed using FlowJo V10.1.

Wang Y., Tang B., Li H., Zheng J., Zhang C., Yang Z., Tan X., Luo P., Ma L., Wang Y., Long L., Chen Z., Xiao Z., Ma L., Zhou J., Wang Y, & Shi C. (2023). A small-molecule inhibitor of Keap1–Nrf2 interaction attenuates sepsis by selectively augmenting the antibacterial defence of macrophages at infection sites. eBioMedicine, 90, 104480.

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Quantifying Antigen-Specific Thymocytes

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Mouse thymi were cut into 8 μm frozen sections using a Leica CM1520 cryostat (Leica Biosystems). Anti-mouse Gr1 and anti-mouse CD45.2 were purchased from BioLegend. Anti-mouse cytokeratin 5, anti-mouse cytokeratin 8, anti-HBsAg, and anti-HBsAg were from Bioss Inc. H-2Ld HBsAg28-39 (IPQSLDSWWTSL) MHC-I tetramer (PE) was used to detect HBsAg-specific CD8⁺ thymocytes in the immunofluorescence (IF) test.

Fang Z., Zhang Y., Zhu Z., Wang C., Hu Y., Peng X., Zhang D., Zhao J., Shi B., Shen Z., Wu M., Xu C., Chen J., Zhou X., Xie Y., Yu H., Zhang X., Li J., Hu Y., Kozlowski M., Bertoletti A, & Yuan Z. (2022). Monocytic MDSCs homing to thymus contribute to age-related CD8+ T cell tolerance of HBV. The Journal of Experimental Medicine, 219(4), e20211838.

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Tumor Immunotherapy Protocol

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Anti-mouse Tim-3 (clone: RMT3-23), anti-mouse PD-1 (clone: J43), anti-mouse Lag-3 (clone: C9B7W), Rat IgG isotype control (clone: 2A3) and polyclonal American hamster IgG were purchased from Bioxcell company for tumor therapy. For Flow cytometry, anti-mouse CD45 (clone: 30-F11), anti-mouse CD4 (clone: GK1.5), anti-mouse CD8a (clone: 53–6.7), anti-mouse Foxp3 (MF23), anti-mouse γδTCR (clone: GL3), anti-mouse CD103 (clone: M290), anti-mouse Tim-3 (clone: 5D12), anti-mouse Lag-3 (clone: C9B7W), anti-mouse Lag-3 (clone: C9B7W), anti-mouse CD206 (clone: MR5D3), anti-mouse CD62L (clone: MEL-14), anti-mouse CD44(clone: IM7), anti-mouse OX40 (OX-86), anti-mouse IL7R (clone: SB/199), anti-mouse GITR (clone:DTA-1) and anti-mouse CD11b (clone: M1/70) were purchased from BD Bioscience. anti-mouse Ki67(clone: SolA15), IFN-γ (clone: XMG1.2), CD11b (M1/70) were purchased from ebioscience. anti-mouse B220 (clone: RA3-6B2), anti-mouse CD8a (clone: 53–6.7), anti-human/mouse Granzyme B (clone: GB11), anti-mouse Granzyme A (clone: 3G8.5), anti-mouse MHC II (clone: M5/114.15.2), anti-mouse Gr-1 (clone: RB6-8C5), anti-mouse CD24 (clone: M1/69), anti-mouse F4/80 (clone: BM8) and anti-mouse NK1.1 (clone: PK136) were purchased from Biolegend. Mitotracker Deep Red FM labeling kit was purchased from Invitrogen. Ghost 510 and Zombie NIR dye was purchased from Tonbo Biosciences and Biolegend.

Yang M., Du W., Yi L., Wu S., He C., Zhai W., Yue C., Sun R., Menk A.V., Delgoffe G.M., Jiang J, & Lu B. (2020). Checkpoint molecules coordinately restrain hyperactivated effector T cells in the tumor microenvironment. Oncoimmunology, 9(1), 1708064.

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Flow cytometric analysis of immune cells

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For cell lines, cells were firstly harvested, centrifuged, and washed in PBS. The cell pellets were resuspended in 100 μL PBS + 1% BSA and stained with fluorescent dye-conjugated antibodies. All antibodies were obtained from Biolegend: anti-human Fas (Cat#305608), anti-mouse/human CD11b (Cat#101206), anti-human HLA-DR (Cat#307609), anti-human CD8 (Cat#344706), anti-mouse Fas (Cat#152607), anti-mouse Gr1 (Cat#108426), anti-mouse CD8 (Cat#100732), and anti-mouse CD45 (Cat#147712). Stained cells were analyzed by flow cytometry. Data files were analyzed using FlowJo.V10 software.

Yang Y., Zhang M., Zhang Y., Liu K, & Lu C. (2023). 5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity. Cancers, 15(5), 1563.

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Murine Fetal Liver Cell Isolation

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E14.5 murine fetal liver cells were incubated with biotin-conjugated anti-mouse CD3𝛆 3 ml/ml, Biolegend, 100304), anti-mouse/human CD11b (3 ml/ml, Biolegend, 101204), anti-mouse CD19 (3 μl/ml, Biolegend, 115504), anti-mouse/human CD45R (3 μl/ml, Biolegend, 103204), anti-mouse GR-1 (3 μl/ml, Biolegend, 108404), anti-mouse CD71 (3 μl/ml, Biolegend, 113803), and anti-mouse Ter119 (5 ml/ml, Biolegend, 116204) for 15 min at 4°C. Cells were washed with PBS containing 2% FBS, 2.5 mM EDTA, and 10 mM glucose. Cells were incubated with MojoSort Streptavidin Nanobeads (75 μl/ml, Biolegend, 480016) for 15 min at 4°C. Cells were washed with P BS containing 2% FBS, 2.5 mM EDTA, and 10 mM glucose. Cells were resuspended in 3 ml PBS containing 2% FBS, 2.5 mM EDTA, and 10 mM glucose and incubated for 6 min with a magnet. Unbound cells were collected.

Tanimura N., Liao R., Wilson G.M., Dent M.R., Cao M., Burstyn J.N., Hematti P., Liu X., Zhang Y., Zheng Y., Keles S., Xu J., Coon J.J, & Bresnick E.H. (2018). GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism. Developmental cell, 46(5), 581-594.e4.

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