Ly6g gr 1

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Ly6G (Gr-1) is a cell surface antigen expressed on mature neutrophils and some myeloid-derived suppressor cells. It is commonly used as a marker for the identification and isolation of these cell types.

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Anti-mouse Ly6G/Gr-1, (2 citations) Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, (2 citations) Anti-Ly6G (Gr-1)-PE (2 citations) α-Ly6G (GR-1, (2 citations)

6 protocols using ly6g gr 1

Isolation and Purification of Pre-B Cells

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C57BL/6 (WT) and Rag1^–/–/VH81X mice were maintained in accordance with local and Home Office rules and ARRIVE guidelines under Project Licence 80/2529.
For each biological replicate, bone marrow from nine 6- to 8-week-old Rag1^−/−/VH81X mice (47 (link), 48 (link)) or from fifteen 12-week-old male wild-type (WT) C57BL/6 mice was incubated with biotinylated antibodies against CD11B (MAC-1; ebioscience), Ly6G (Gr-1; ebioscience), Ly6C (Abd Serotec), TER119 (ebioscience), and CD3E (ebioscience) followed by incubation with streptavidin MACs beads (Miltenyi), to deplete macrophages, granulocytes, erythroid lineage, and T cells. Pre-B cells (surface IgM⁻CD25⁺B220⁺CD19⁺) were then flow sorted on a BD FACSAria in the Babraham Institute Flow Cytometry facility. Antibodies used were CD45R BV421 (B220, RA3-6B2, Biolegend), CD19 PerCP-Cy5.5 (1D3, BD Pharmingen), CD25 APC (PC61.5, eBioscience), and IgM PE (eB121-15F9, eBioscience). Sort purities were all greater than 92%.

Matheson L.S., Bolland D.J., Chovanec P., Krueger F., Andrews S., Koohy H, & Corcoran A.E. (2017). Local Chromatin Features Including PU.1 and IKAROS Binding and H3K4 Methylation Shape the Repertoire of Immunoglobulin Kappa Genes Chosen for V(D)J Recombination. Frontiers in Immunology, 8, 1550.

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Multiparameter Flow Cytometry Analysis

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Single cell suspensions from lungs and spleens were prepared by chopping tissue into small pieces followed by digestion with Liberase DL (Roche) at 37°C for 30 min. RBC were removed by treatment with ACK lysis buffer followed by washing with PBS pH 7.4. Cells were treated with fixable viable dye (eBiosciences) for 30 min at 4°C in the dark. Fc receptors were blocked with anti-CD16/32 for 10 min at 4°C in the dark. Finally, cells were resuspended in staining buffer (PBS pH 7.4 containing 0.1% BSA and 0.01% NaN₃) and stained at saturating concentrations of antibodies specific for CD11b, #83-0112; CD45, #86-0451; Ly-6C, #48-5932; Ly-6G (GR-1), #17-9668; and CD4, #83-0042 (eBiosciences). Samples were fixed in 2% paraformaldehyde solution for 20 min and data collected using an LSRII flow cytometer (Becton-Dickenson) and analyzed using Winlist Software (Verity Software House, Inc.), FlowJo Software (Tree Star, Inc.).

Reddy V.P., Chinta K.C., Saini V., Glasgow J.N., Hull T.D., Traylor A., Rey-Stolle F., Soares M.P., Madansein R., Rahman M.A., Barbas C., Nargan K., Naidoo T., Ramdial P.K., George J.F., Agarwal A, & Steyn A.J. (2018). Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection. Frontiers in Immunology, 9, 860.

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Assessing Immune Cell Apoptosis and Oxidative Stress

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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies, and an Annexin V-FITC apoptosis kit were purchased from eBioscience (San Diego, CA, USA). The 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), α-lipoic acid, and 5-methoxytryptamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3534 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-γH2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA).
The 5-methoxytryptamine-α-lipoic acid (MLA) was synthetized using α-lipoic acid and 5-methoxytryptamine at the drug department of the Institute of Radiation Medicine, CAMS. Melatonin (MLT) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). MLA and MLT were dissolved in a 5% carboxymethyl cellulose (CMC) solution.

Li D., Tian Z., Tang W., Zhang J., Lu L., Sun Z., Zhou Z, & Fan F. (2016). The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury. International Journal of Molecular Sciences, 17(6), 935.

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Isolation and Characterization of Pro-B and Pre-B Cells

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Bone marrow was flushed from 12–15 12-week (young) or 19–22 month (aged) healthy male C57BL/6 mice per replicate and depleted of macrophages, granulocytes, erythroid lineage, and T cells using biotinylated antibodies against Cd11b (MAC-1; ebioscience), Ly6G (Gr-1; ebioscience), Ly6C (Abd Serotec), Ter119 (ebioscience), and Cd3e (ebioscience) followed by streptavidin MACs beads (Miltenyi). Thereafter, pro-B (B220⁺CD19⁺CD43⁺CD25⁻IgM⁻) and pre-B (B220⁺CD19⁺CD43^-CD25⁺IgM⁻) cells were flow sorted on a BD FACSAria in the Babraham Institute Flow Cytometry facility. Antibodies used were CD45R BV421 (B220, RA3-6B2, Biolegend), CD19 PerCP-Cy5.5 (1D3, BD Pharmingen), CD43 FITC (S7, BD Pharmingen), CD25 APC (PC61.5, eBioscience), and IgM PE (eB121-15F9, eBioscience). Purities were > 85% for pro-B and > 90% for pre-B.
The flow cytometry data were quality checked using the automated method in FlowAI [63 (link)] and low-quality events were filtered out. Lymphocytes, singlets, and B220 positive cells were gated using FlowJo V10.1 software (FlowJo, LLC) and all compensated parameters were exported for subsequent analysis. Target populations were also manually gated and exported. A tSNE plot was constructed using the Rtsne package with default parameters [64 ]. To identify populations of interest, manual gates were projected onto the tSNE plot.

Koohy H., Bolland D.J., Matheson L.S., Schoenfelder S., Stellato C., Dimond A., Várnai C., Chovanec P., Chessa T., Denizot J., Manzano Garcia R., Wingett S.W., Freire-Pritchett P., Nagano T., Hawkins P., Stephens L., Elderkin S., Spivakov M., Fraser P., Corcoran A.E, & Varga-Weisz P.D. (2018). Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells. Genome Biology, 19, 126.

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Multiparameter Immune Cell Analysis

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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies were purchased from eBioscience (San Diego, CA, USA). The 21, 71-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich (St Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3434 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-H2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA). Hei brick tea was purchased from Baishaxi Tea Industry (Hunan, China). Malondialdehyde (MDA), SOD, CATand GSHPx reagent kits were purchased from Nanjing Jiancheng Bioengineering (Jiangsu, China).

Long W., Zhang G., Dong Y, & Li D. (2018). Dark tea extract mitigates hematopoietic radiation injury with antioxidative activity. Journal of Radiation Research, 59(4), 387-394.

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Anti-inflammatory Activities of Cell Signaling

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DMEM medium and fetal bovine serum (FBS) were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). Lipopolysaccharide (LPS) isolated from Escherichia coli 0111:B4 and dexamethasone were purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF-α and IL-6 ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA). The antibodies against inducible NO synthase (iNOS), COX-2, IκB-α, phospho-p38, phospho-JNK, phospho-ERK, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Ly-6G (Gr-1) was purchased from eBioscience (San Diego, CA, USA). All other reagents were of the highest grade commercially available.

Kim Y.S., Hwang J.W., Jang J.H., Son S., Seo I.B., Jeong J.H., Kim E.H., Moon S.H., Jeon B.T, & Park P.J. (2016). Trapa japonica Pericarp Extract Reduces LPS-Induced Inflammation in Macrophages and Acute Lung Injury in Mice. Molecules, 21(3), 392.

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