Gr1 apc cy7

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The Gr1-APC-Cy7 is a fluorescently labeled antibody that binds to the Gr1 antigen. It is designed for use in flow cytometry applications.

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APC-Cy7 (106 citations) Gr1-APC-Cy7 (RB6-8C5, (4 citations)

8 protocols using gr1 apc cy7

Multiparametric Flow Cytometry for Immune Profiling

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Flow cytometry was performed using a BD FACSCantoII™ flow cytometer and analysis run with FACSDiva 6.1.1 software (Becton–Dickinson). Cells were stained with anti-mouse CD45-PB (#103125), CD11b-PE/Cy5 (#101209), F4/80-APC (#123115), F4/80-FITC (#123107), CD206-FITC (#141703), CD80-PE (#104707), SiglecF1-PE (#142403), Gr1-APC/Cy7 (#108423), CD3-APC (#100235), CD4-PE (#100407), CD8-PE/Cy5 (#100709) (BioLegend, San Diego, CA, USA) for 20 min at 4 °C, washed and resuspended in FACS Buffer (PBS, 5 mM EDTA and 0.2% BSA).
Intracellular staining for Ki67-AF488 (#561165, BD Biosciences) was performed by adding 3 ml cold 70% ethanol to the cell pellet and incubating at − 20 °C for 1 h, followed by staining with the antibody at RT for 30 min.

Tomay F., Marinelli A., Leoni V., Caccia C., Matros A., Mock H.P., Tonelli C, & Petroni K. (2019). Purple corn extract induces long-lasting reprogramming and M2 phenotypic switch of adipose tissue macrophages in obese mice. Journal of Translational Medicine, 17, 237.

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Multiparameter Flow Cytometry of Tumor Samples

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Flow cytometric analysis was performed as described previously (25 (link)). Briefly, tumor tissues were cut into pieces and digested with DMEM containing hyaluronidase (1.5 mg/ml, Sigma-Aldrich, USA), collagenase type 1A (1.5 mg/ml, Sigma-Aldrich, USA), and deoxyribonuclease I (20 U/ml, Sigma-Aldrich, USA) at 37°C for 45 min. The tissue mixture was passed through a 70-µm nylon cell strainer to make single cell suspension, which was then washed and resuspended in cold flow buffer (1% bovine serum albumin and 0.1% NaN₃ in PBS). After being blocked with a rat anti-mouse CD16/CD32 antibody (BD Pharmingen, USA), the following fluorochrome-conjugated anti-mouse antibodies were used: CD45-BV421, CD11b-BV510, CD8a-PE-Cy7, CD4-PE, CD25-APC, NK1.1-APC-Cy7, F4/80-FITC, CD206-PE-Cy7, CD11c-APC, and Gr-1-APC-Cy7 (all from BioLegend, USA). 7-Amino-actinomycin D (7AAD) (eBioscience, USA) was used as a viability dye to exclude dead cells. Flow cytometric data were collected using a Gallios flow cytometer (Beckman, USA) and analyzed using the Kaluza software (version 1.3).

Fan P., Qiang H., Liu Z., Zhao Q., Wang Y., Liu T., Wang X., Chu T., Huang Y., Xu W, & Qin S. (2022). Effective low-dose Anlotinib induces long-term tumor vascular normalization and improves anti-PD-1 therapy. Frontiers in Immunology, 13, 937924.

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Tumor Immune Cell Profiling

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Mice-bearing tumors were intracardially perfused with PBS. Tumor tissues were isolated and single cell suspensions were prepared by using the digesting DMEM medium containing collagenase type 1A (200 U/ml), hyaluronidase (1000 U/ml) and DNase (20 U/ml). Rat anti-mouse CD16/CD32 monoclonal antibody was added into the single-cell suspensions before other antibody staining. After staining, cells were washed with cold flow buffer (1% BSA, 0.1% NaN₃ in PBS), and 7AAD reagent (eBioscience) was added (5 μl/tube) prior to running the flow analysis. Flow cytometry data were acquired on a Gallios flow cytometer (Beckman) and analyzed with a Kaluza software (version 1.3). The following fluorescence-labeled or isotype-matched anti-mouse antibodies were used: CD8-FITC, CD4-PE, CD69-PE-Cy7, CD44-APC, CD62L-APC-Cy7, CD4-Alexa Flour 700, Gr1-APC-Cy7, CD25-APC-Cy7, Ly6G-FITC, F4/80-PE, CD45-BV421, and CD11b-BV510 (BioLegend) [8 (link), 17 (link)].

Xu H., Qi Z., Zhao Q., Xue J., Zhu J., He Y., Liu G, & Qin S. (2022). Lentinan enhances the antitumor effects of Delta-like 1 via neutrophils. BMC Cancer, 22, 918.

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Tumor Immune Cell Profiling by Flow Cytometry

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Tumor tissues were harvested and digested in DMEM with collagenase type 1A (1.5 mg/ml), hyaluronidase (1.5 mg/ml) and DNase (20 U/ml) at 37°C for 45 minutes. After digestion, tissues were passed through 70 μm cell strainers to obtain single cell suspensions. Single-cell suspensions were then incubated with a rat anti-mouse CD16/CD32 monoclonal antibody to block Fcγ receptors. After blocking, the cells were incubated with a flow cytometric antibody cocktail for 15 minutes in a refrigerator in the dark. The following antibodies were used for flow cytometry analysis: CD4-PE (Clone: GK1.5, catalog 100408), CD8a-FITC (Clone: 53-6.72, catalog 100706), CD4-Alexa Flour 700 (Clone: RM4-4, catalog 116022), PD-L1-PE (Clone: 10F.9G2, catalog 124308), CD44-APC (Clone: IM7, catalog 103012), CD69-PE-Cy7 (Clone: H1.2F3, catalog 104512), CD62L-APC-Cy7 (Clone: MEL-14, catalog 104428), F4/80-FITC (Clone: BM8, catalog123108), Gr1-APC-Cy7 (Clone: RB6-8C5, catalog108424), CD45-BV421 (Clone: 30-F11, catalog 103134), and CD11b-BV510 (Clone: M1/70, catalog 101263) (from BioLegend) [37 (link), 38 (link)]. The reagent 7-aminoactinomycin D (7AAD, eBioscience) was added to stain dead cells (5 μl/tube) just before flow cytometry analysis. Samples were analyzed on a Gallios flow cytometer (Beckman), and data were analyzed with Kaluza software (version 1.3).

Zhang Y., Gao J., He Y., Qi Z., Qian L., Chen W., Xu H., Yue Y., Mao X., Guo S., Zhou Y., Zhou S., Qin S., Zhang X, & Huang Y. (2023). Vascular Normalization Was Associated with Colorectal Tumor Regression upon Anti-PD-L1 Combinational Therapy. Journal of Immunology Research, 2023, 5867047.

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Phagocytic Capacity of H. influenzae

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A flow cytometric method was used to determine the phagocytic capacity of H. influenzae by neutrophils and alveolar macrophages. FITC labeled H. influenzae was adjusted to 1 × 10⁹ CFU/ml and added to resuspended BALF cells (1 × 10⁷/ml). After incubated at 37 °C for 10 minutes, cells were washed with PBS to remove potentially excessive extracellular bacteria and subsequently stained with the following fluorochrome-labeled antibodies (all obtained from Biolegend, USA): PerCP/Cy5.5-CD45, PE-F4/80 and APC-Cy7-Gr1. The cells were incubated with these antibodies for 30 minutes at 4 °C in the dark. Then they were washed, resuspended with PBS and detected using a Beckman Coulter (Galios, USA) flow cytometry. Among all the cells positive for CD45, neutrophils were defined by positive staining for Gr1 and alveolar macrophage were defined by positive staining for F4/80. Phagocytosing cells were those positive for H. influenzae-FITC. Phagocytic capacity was evaluated by MFI of FITC. Flow cytometry data were analysed with Flowjo software (version 10.0.7).

Yang X., Wang Y., Zhao S., Wang R, & Wang C. (2018). Long-term exposure to low-dose Haemophilus influenzae during allergic airway disease drives a steroid-resistant neutrophilic inflammation and promotes airway remodeling. Oncotarget, 9(38), 24898-24913.

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Multiparametric Immune Cell Profiling

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Splenocytes and synoviocytes were blocked with mixture of supernatant of anti-CD16/32 mAb (2.4G2) and 5% heat inactivated rat serum. Then the cells were stained with two sets of cocktail mAbs labelled with fluorescent dyes. Set 1 consists of FITC-anti-Ly-6G/Ly-6C (Gr-1) (RB6-8C5) (BD biosciences), PE-anti-CD3ε (145-2C11) (Tonbo Bioscience, Burlingame, California, USA), PE-Cy7-anti-TER119, APC-anti-CD11c (N418), APC-Cy7-anti-CD19 (6D5) (BioLegend), eFlour450-anti-CD4 (GK1.5) (Tonbo Bioscience) mAbs. Set 2 consists of PerCP-Cy5.5-labelled anti-CD3ε, -CD19, -NK1.1 (PK136) (Tonbo Bioscience), -TER119 (eBioscience, San Diego, California, USA), PE-SiglecF (BD Biosciences), APC-F4/80 (eBioscience), APC-Cy7-Gr-1 and Pacific Blue-CD11b (BioLegend). The stained samples were analysed with FACSCanto II with FACSDiva software (BD Biosciences). Details to define cellular subsets are indicated in online supplementary figure 2. Cells were suspended in 0.4% Trypan Blue and total viable cell numbers were counted using Bürker-Türk haemocytometer under a microscope. With total viable cell numbers and frequencies obtained by flowcytometry (online supplementary figure 3), the cell numbers of a certain cell-lineage in the spleen or synovium were calculated.

Yahagi A., Saika T., Hirano H., Takai-Imamura M., Tsuji F., Aono H., Iseki M., Morita Y., Igarashi H., Saeki Y, & Ishihara K. (2019). IL-6-PAD4 axis in the earliest phase of arthritis in knock-in gp130F759 mice, a model for rheumatoid arthritis. RMD Open, 5(2), e000853.

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Monocyte-derived Macrophage Phenotyping

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Bone marrow cells were flushed from femurs and tibias, centrifuged, then exposed to geys buffer for 5 min on ice, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with antibodies for 20 min at 4 °C, and washed before sorting CD115+/CD11c-/NK- monocytes using an Influx cell sorter (BD) [68 (link)]. Monocytes were cultured in complete RPMI medium (Thermo Fisher Scientific) and exposed to 100 ng/mL CSF1, 50 µM Q-VD-OPh and 50 µM Ac-YVAD-cmk. After five days, macrophages were studied by flow cytometry using AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BUV737-CD43 (#564398, BD). For the sorting of monocytes, the antibodies used were the following: APC-CD11b (#17-0112-83, Thermo Fisher Scientific), PE/Cy7-CD11c (#561022, BD), FITC-NK (#553164, BD), PerCP/Cy5.5-Ly6C (#560525, BD), AlexaFluor700-Ly6G (#561236, BD), biotin-CD115 (#135507, Biolegend), PE-Streptavidin (#554061, BD). The data were analyzed with FlowJo software v. 10.0.00003.

Solier S., Mondini M., Meziani L., Jacquel A., Lacout C., Berghe T.V., Julé Y., Martinou J.C., Pierron G., Rivière J., Deloger M., Dupuy C., Slama-Schwok A., Droin N., Vandenabeele P., Auberger P., Deutsch E., El-Benna J., Dang P.M, & Solary E. (2023). Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues. International Journal of Molecular Sciences, 24(4), 4151.

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Interstitial Macrophages Identification

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Right lungs were digested with the Lung Dissociation kit (Miltenyi Biotec), filtered, erythrocytes were removed using ACK and nucleated cells were collected. Cells were washed with ice-cold PBS, incubated with Fc block (Murine TruStain FcX, Biolegend, 1/50 dilution) for 15 min, incubated with the antibodies for 20 min at 4 °C, washed, then fluorescence was measured with a BD LSRFortessa X-20. Antibodies used were the following: AlexaFluor700-F4/80 (#MCA497A700, Bio-Rad), FITC-CD45 (#103108, Biolegend), BV510-CD11b (#101245, Biolegend), PerCP/Cy5.5-CD11c (#560584, BD), APC/Cy7-GR1 (#108424, Biolegend), PE-CF594-SiglecF (#562757, BD), PE-CD71 (#113808, Biolegend), PE/Cy7-CD206 (#141720, Biolegend), APC-CD54 (#116120, Biolegend), PB-CD40 (#124626, Biolegend), BV711-CD64 (#139311, Biolegend), BV605-IA-IE (#563413, BD), BV650-CD24 (#563545, BD), BUV737-CD43 (#564398, BD). The data were analyzed with FlowJo software v. 10.0.00003. Interstitial macrophages were selected according to their larger size (FSC) and granularity (SSC), on CD45 expression to select leukocytes and GR1-positive cells will be excluded to remove neutrophils, they are equally CD11b high, SiglecF negative, IA-IE positive and CD24 negative.

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