Gr 1 clone rb6 8c5

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Gr-1 (clone RB6-8C5) is a laboratory reagent produced by Thermo Fisher Scientific. It is a monoclonal antibody that can be used to identify and quantify granulocytes in various biological samples. The core function of this product is to serve as a tool for cellular analysis and research applications.

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RB6-8C5 (38 citations) Gr-1 (RB6-8C5) (23 citations) Anti-Gr-1 (clone RB6–8C5, (5 citations) (clone RB6-8C5, (3 citations) Ly-6G/Gr-1 (clone RB6-8C5, (3 citations) Gr-1-APC (Clone RB6-8C5, (3 citations) PE-Cy7-anti-Gr-1 (clone RB6–8C5, (2 citations) Ly6G (Gr-1) PE Cy7-conjugated (clone RB6-8C5, (2 citations) Anti-GR-1 PE mAb (clone RB6-8C5; (2 citations)

7 protocols using gr 1 clone rb6 8c5

Characterization of Tumor-Infiltrating Lymphocytes

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Single cell suspensions from spleens and lymph nodes were prepared as described.6 (link) TIL were isolated from pooled tumors as described.26 (link) All flow cytometry experiments were performed at least 3 times. Single cell suspensions were incubated with mouse Fc receptor binding inhibitor for 10 minutes before staining with antibodies to CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD19 (clone eBio1D3), CD86 (clone GL1), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), CD69 (clone H1.2F3), CD44 (clone IM9), CD62L (clone MEL-14), and CD11c (clone N418; all from eBioscience, San Diego, CA) for 30 minutes. Flow cytometry was performed using FACS Calibur (BD Biosciences) and the lymphocyte population was selected by gating CD45⁺ cells. The data were analyzed using Flow Jo software (Tree Star, Ashland, OR). For tetramer staining, 10 μL of PE labeled HLA-A*02:01 Human HPV16 E7 tetramer (NIH Tetramer Core Facility) was added to 200 μL mouse lymphocyte suspension (1×106 cells per tube). After incubation for 30 minutes, cells were centrifuged and resuspended in phosphate-buffered saline with 1% paraformaldehyde and then analyzed by flow cytometry. PE labeled HLA-A*02:01 human mesothelin tetramer (NIH Tetramer Core Facility) was used as control.

Dai M., Hellstrom I., Yip Y.Y., Sjögren H.O, & Hellstrom K.E. (2018). Tumor Regression and Cure Depends on Sustained Th1 Responses. Journal of Immunotherapy (Hagerstown, Md. : 1997), 41(8), 369-378.

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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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Cytokine profiling of obese WT and IFNAR-/- mice

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eWAT and liver was isolated from obese WT and IFNAR^−/− mice. eWAT-isolated SVF cells and liver immune cells were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (1 μg/ml; Calbiochem). Briefly, cells were stained with Live/dead stain (Zombie UV Dye; 1:250; Biolegend), B220 (clone RA3-6B2; 1:100; Biolegend), CD45 (clone 104; 1:500), CD11b (clone M1/70; 1:100), F4/80 (clone BM8; 1:100), Gr1 (clone RB6-8C5; 1:100), CD4 (clone GK1.5; 1:50), CD8 (clone 53-6.7; 1:100), IFNγ (clone XMG1.2; 1:100) TNF (clone MP6-XT22; 1:100), and IL-6 clone (MP5-20F3; 1:100) (all ebioscience). Data were collected using a LSR Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Chan C.C., Damen M.S., Moreno-Fernandez M.E., Stankiewicz T.E., Cappelletti M., Alarcon P.C., Oates J.R., Doll J.R., Mukherjee R., Chen X., Karns R., Weirauch M.T., Helmrath M.A., Inge T.H, & Divanovic S. (2020). Type I interferon sensing unlocks dormant adipocyte inflammatory potential. Nature Communications, 11, 2745.

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Multiparametric Flow Cytometric Analysis of HSCs

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Cells were surface-stained in PEB buffer (PBS supplemented with 0.5% BSA and 2mM EDTA) for 20–30 min on ice. Multiparametric flow cytometric analyses were performed on a LSRII equipped with FACS Diva 6.1 software (BD Biosciences) and analysed with FlowJo software (Tree Star). Dead cells were excluded by FSC, SSC and 4’,6-diamino-2-phenylindole (DAPI, Sigma) staining. Cell sorting experiments were performed on Aria Cell Sorter (BD Biosciences). HSCs measured by flow cytometry were defined as: Lineage⁻ Sca1⁺ cKit⁺ Flt3⁻. Antibodies used: anti-mouse mABs Sca1/Ly-6A/E (clone D7, ebioscience), CD117/cKit (clone ZB8, Biolegend), CD135/Flt3 (clone AZF10, ebioscience), lineage cocktail (BD Pharmingen cat# 559971), CD41 (clone RMV-7, ebiosicence), CD45 (clone 30-F11, ebioscience), Ter119 (clone TER-119, ebioscience), CD31 (clone MEC13.3, ebioscience), Gr-1 (clone RB6-8C5, ebioscience), CD11b (M1/70, ebioscience), CD115/c-fms (clone AFS98, ebioscience), F4/80 (BM8, ebioscience), and BrdU (clone BU20A, BD Pharmingen with BrdU flow kit reagents for fixation and permeabilization).

Pierce H., Zhang D., Magnon C., Lucas D., Christin J., Huggins M., Schwartz G.J, & Frenette P.S. (2017). Cholinergic signals from the CNS regulate G-CSF-mediated HSC mobilization from bone marrow via a glucocorticoid signaling relay. Cell stem cell, 20(5), 648-658.e4.

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Isolation and Analysis of Mouse Hematopoietic Stem Cells

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Cells were analyzed using LRSII (BD, Pharmingen) and sorted using FACS-ARIA II (BD, Pharmingen). The following antibodies were used: anti-CD45 FITC (clone 30-F11 Biolegend), anti-CD31 FITC (clone MEC13.3 Biolegend), anti-Ter119 FITC (clone TER-119 Biolegend), anti-Sca1 Pacific Blue (clone D7 Biolegend), anti-CD51 PE (clone RMV-7 Biolegend), bio-Lineage panel antibodies [CD4 (clone GK1.5 eBioscience), CD8 (clone 53-6.7 eBioscience), CD3 (clone 145-2C11 eBioscience), Ter119 (clone TER-119 eBioscience), CD11b (clone M1/70 eBioscience), Gr1 (clone RB6-8C5 eBioscience), NK1.1 (clone PK136 eBioscience), B220 (clone RA3-6B2 eBioscience)], anti-ckit APC (clone 2B8 eBioscience), anti-Sca1 PE (clone D7 eBioscience), anti-CD34 FITC (clone RAM-34 eBioscience), anti-SLAM (CD150) PerCP cy5.5 (clone TEC15-12F12.2 Biolegend), anti-Cd11b APC (clone M1/70 Biolegend), and anti-Gr1 PE (clone RB6-8C5 Biolegend).

Guarnerio J., Mendez L.M., Asada N., Menon A.V., Fung J., Berry K., Frenette P.S., Ito K, & Pandolfi P.P. (2018). A non-cell-autonomous role for Pml in the maintenance of leukemia from the niche. Nature Communications, 9, 66.

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Identification and Quantification of Muscle Macrophages

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Single-cell suspensions from muscle were prepared by collagenase II (Worthington Biochemical Corp, Lakewood, NJ, USA) digestion followed by staining with fluorochrome-conjugated antibodies.
Antibodies used: (1) Gr1 Clone RB6-8C5 (eBioscience, ThermoFisher, Waltham, MA, USA, Cat#48-5931-82); (2) F4/80 Clone BM8 (Invitrogen, Cat# 11-4801-82); (3) CD11b Clone M1/70 (Invitrogen, ThermoFisher, Waltham, MA, USA, Cat#17-0112-81); (4) Siglec-F Clone E50-2440 (RUO), (BD BioSciences, ThermoFisher, Waltham, MA, USA, Cat# 552126); (5) CD206 Clone C068C2 (BioLegend, San Diego, CA, USA, Cat#141701) and live dead stain (eBioscience). Data acquisition was performed on a FACS Aria or Fortessa (BD Biosciences). UltraComp eBeads (Cat#01333342, Invitrogen) were used to generate single-stain controls. Data was analyzed using FlowJo software (version 10.2) and gating strategies as previously described [39 (link)] to discriminate against dead cells, debris, and doublets were utilized. M1 macrophages were defined as Gr1^low-med, F4/80⁺, and CD11b⁺.

Lynch C.A., Acosta S.A., Anderson D.M., Rogers G.E., Wilson-Rawls J, & Rawls A. (2024). The Transcription Factor Mohawk Facilitates Skeletal Muscle Repair via Modulation of the Inflammatory Environment. International Journal of Molecular Sciences, 25(9), 5019.

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Liver Nonparenchymal Cell Isolation and Characterization

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Liver NPCs were isolated from sham or IR livers, as described above. We incubated 1×10⁶ cells first with rat anti–mouse CD16/32 for 10 minutes, followed by staining with rat anti–mouse F4/80 (clone BM8), CD11b (clone M1/70), Gr1 (clone RB6-8C5), TIM-4 (clone RMT4-54), MerTK (clone DS5MMER), or isotype-matched control Ab (eBioscience) for 20 minutes. Cells were washed with PBS and subjected to ﬂow cytometry analysis with BD LSR Fortessa (BD Biosciences). A representative FACS plot of liver NPCs at day 3 after reperfusion is shown in Supplemental Figure 4. Clearly, KCs expressed higher levels of MerTK than iMΦs.

Zhang H., Ni M., Wang H., Zhang J., Jin D., Busuttil R.W., Kupiec-Weglinski J.W., Li W., Wang X, & Zhai Y. (2023). Gsk3β regulates the resolution of liver ischemia/reperfusion injury via MerTK. JCI Insight, 8(1), e151819.

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