Pecy7 gr 1

Manufactured by BioLegend

Sourced in United States

The PEcy7-Gr-1 is a fluorochrome-conjugated antibody that binds to the Gr-1 antigen. Gr-1 is a marker that is expressed on granulocytes, including neutrophils, eosinophils, and myeloid-derived suppressor cells. The PEcy7 fluorochrome allows for detection and analysis of Gr-1-positive cells using flow cytometry.

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6 protocols using pecy7 gr 1

Immunomodulation Pathway Evaluation

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Dopamine, SCH58261, Collagenase A, 2′,7′‐Dichlorofluorescein diacetate (H2DCF), and Coformycin (Erythro‑9‑(2‑hydroxy‑3‑nonyl) adenine) were purchased from Sigma‐Aldrich. 4‐bromomethyl phenylboronic acid (BPBA), Amino polyethylene glycol (PEG), Rhodamine B isothiocyanate (Rhodamine B), and Fluorescein5(6)‐isothiocyanate (FITC) were acquired from Aladdin Industrial Corporation. Bodipy and Live/Dead staining kits were obtained from Thermo Fisher Scientific. Adenosine, CCK‐8, FITC‐Annexin V Apoptosis Detection Kit, Enhanced ATP Assay Kit, BCA assay kit, and Protease inhibitor cocktail for general use (100X) were purchased from Beyotime Biotechnology. Percp‐CRT antibody was purchased from StressMarq Biosciences Inc. Antibodies of APC‐CD73, Alexa Fluor 488‐HMGB1, CD16/32, Foxp3, APC‐CD11c, FITC‐CD80, PE‐CD86, PE‐CD40, FITC‐MHCII, FITC‐CCR7, PE‐CD45, PEcy7‐Gr‐1, APC‐CD11b, APC‐CD3, FITC‐CD4, PEcy7‐CD8α, and PE‐IFNγ were purchased from BioLegend. The antibody of A2AR was obtained from Proteintech Group, Inc. Adenosine ELISA Kit was obtained from Shanghai Jianglai Biological Technology Co., Ltd. ELISA kits of TNF‐α, IL‐6, IFN‐γ, and IL‐12p40 were purchased from Dakewe Biotech Co., Ltd. Cytokines of GM‐CSF, IL‐4, and IL‐2 were purchased from Shanghai Jinan Technology Co., Ltd. DNase I and Bouin's solution were purchased from Beijing solarbio science&technology co., ltd.

Liu Y., Liu Y., Xu D., Zang J., Zheng X., Zhao Y., Li Y., He R., Ruan S., Dong H., Gu J., Yang Y., Cheng Q, & Li Y. (2022). Targeting the Negative Feedback of Adenosine‐A2AR Metabolic Pathway by a Tailored Nanoinhibitor for Photothermal Immunotherapy. Advanced Science, 9(14), 2104182.

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Tumor-Infiltrating Lymphocyte Isolation and Characterization

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TILs isolation was done as previously described [21 ]. TDLNs were processed, suspended in RPMI-1640, then washed and filtered. Tumor cells or TILs were blocked with anti-mouse CD16/32 (FcR blocker; BioLegend, Cat#101302) for 10 min at room temperature, and then stained with Pacific blue-CD45 (BioLegend, Cat#103126), APC/Cy7-CD4 (BioLegend, Cat#100414), PerCP/Cy5.5-CD8a (BioLegend, Cat#100734), PE-CD11c (BioLegend, Cat#117308), FITC-F4/80 (BioLegend, Cat#123108), APC/Cy7-CD11b (BioLegend, Cat#101226), and PE/Cy7-Gr-1 (BioLegend, Cat#108416) at room temperature for 30 min. For intracellular staining cells were fixed and permeabilized according to the manufacturer’s instructions (eBioscience, Cat#00-5523-00) and stained with PE-FOXP3 (eBioscience, Cat#12-5773-82) and Alexa Fluor 647-IDO1 (BioLegend, Cat#654004). All samples were analyzed with LSRII flow cytometer and FlowJo software (version 10.0.7).

Li A., Barsoumian H.B., Schoenhals J.E., Cushman T.R., Caetano M.S., Wang X., Valdecanas D.R., Niknam S., Younes A.I., Li G., Woodward W.A., Cortez M.A, & Welsh J.W. (2018). Indoleamine 2,3-dioxygenase 1 inhibition targets anti-PD1-resistant lung tumors by blocking myeloid-derived suppressor cells. Cancer letters, 431, 54-63.

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Tumor-Infiltrating Cell Profiling by Flow Cytometry

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Fresh mouse tumor tissues were dissociated into single-cell suspensions with mouse tumor dissociation kits (Miltenyi Biotec) following the manufacturer's instructions. After lysis of red blood cells, the cells were centrifuged, suspended in FACS buffer, and incubated with anti-mouse CD16/32 for 15 min to avoid non-specific binding. Then cells were stained for 45 min at 4 °C in the dark. The cells were washed once with PBS, suspended in 200 μL PBS, and analyzed by BD FACS Aria II (BD Biosciences, San Jose, CA). For detection of tumor-infiltrated cells, cell staining included the following antibodies: PE-CD11b, APC-CD4, and PE-cy7-CD8 from eBioscience; PE-cy7-Gr1, BV421-F4/80, and APC-human CCR2 from Biolegend (San Diego, CA, USA); and APC-conjugated mouse CCR2 from R&D (Minneapolis, MN, USA). A minimum of 10,000 events were acquired for each sample.

Yao W., Ba Q., Li X., Li H., Zhang S., Yuan Y., Wang F., Duan X., Li J., Zhang W, & Wang H. (2017). A Natural CCR2 Antagonist Relieves Tumor-associated Macrophage-mediated Immunosuppression to Produce a Therapeutic Effect for Liver Cancer. EBioMedicine, 22, 58-67.

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Multiparametric Flow Cytometry Analysis

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The cells were blocked in 10% rat serum for 30 minutes on ice before they were washed with PBS and centrifuged at 600 ×g for 5 minutes at 4°C. The cells were resuspended in FACS staining buffer and stained with combinations of PE-CD11b (BD Biosciences, clone M1/70), PerCPCy5.5-CD45 (BD Biosciences, clone 30-F11), APC-CD3 (BD Biosciences, clone 145-2C11), PE-Cy7-Gr1 (Biolegend, clone RB6-8C5), PE-Cy7-CD11c (BD Biosciences, clone HL3), and 647-MHC class II (BD Biosciences, clone M5/114.15.2) antibodies for 30 minutes on ice. Finally, the cells were washed in PBS, centrifuged at 600 ×g for 5 minutes at 4°C, and resuspended in FACS buffer before they were run on a FACSverse flow cytometer, and 10⁶ events were acquired per sample using forward scatter (FSC) and side scatter (SSC). The analysis was performed using the FACSuite software [25 (link)]. Positive staining was determined based on the respective isotype controls and the respective fluorescent minus one (FMO) controls. The mean fluorescent intensity (MFI) was calculated as the geometric mean of each population in the CD45 and CD11b gates, respectively [25 (link)].

Ellman D.G., Degn M., Lund M.C., Clausen B.H., Novrup H.G., Flæng S.B., Jørgensen L.H., Suntharalingam L., Svenningsen Å.F., Brambilla R, & Lambertsen K.L. (2016). Genetic Ablation of Soluble TNF Does Not Affect Lesion Size and Functional Recovery after Moderate Spinal Cord Injury in Mice. Mediators of Inflammation, 2016, 2684098.

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Isolation and Immunophenotyping of Mouse Hematopoietic Cells

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Bone marrow and spleen of a transplant recipient mouse were harvested, and single cell suspensions were prepared as previously described34 (link). Briefly, the femurs and tibias were dissected using sterile technique, flushed with PBS/5% FBS, and cells were passed through a 70 μm strainer. Spleens were dissected cleanly using sterile technique, placed on a 70 μm strainer and using the plunger end of a syringe, gently passed through the strainer. Red blood cells were lysed using RBC lysis buffer (Biolegends, San Diego, California). Cells were resuspended in PBS/5% FBS, and then counted using a Cellometer Mini cell counter (Nexcelom, Lawrence, MA). One million cells of each sample were then distributed into microcentrifuge tubes and labelled with fluorochrome-conjugated antibodies APC/Cy7-B220, FITC-Ter119, PE/Cy7-Gr1, and PerCP/Cy5.5-CD11b (all antibodies were purchased from Biolegends and added at predetermined optimum concentrations). Immunophenotyping was performed in the UVA Flow Cytometry core lab using a Fortessa cytometer, and data were analyzed using the FlowJo program (FlowJo LLC, Ashland, Oregon).

Hu Y., Belyea B.C., Li M., Göthert J.R., Gomez R.A, & Sequeira-Lopez M.L. (2017). Identification of cardiac hemo-vascular precursors and their requirement of sphingosine-1-phosphate receptor 1 for heart development. Scientific Reports, 7, 45205.

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Isolation and Characterization of Murine Hematopoietic Stem and Progenitor Cells

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Bone marrow cells were isolated by flushing one femur and tibia with 3 mL Ca2+ and Mg2+ free HBSS supplemented with 2% HI FBS, filtered through a 50 μm mesh and counted using a hemocytometer. Splenocytes were obtained by mashing between two glass slides and filtered through a 100 μm mesh. Lineage cocktail was comprised of biotin labeled lineage panel (CD3e 145-2C11, B220 RA3-6B2, TER119, Gr-1 RB6-8C5, Mac1 M1/70 eBioscience) along with biotin CD4 (GK1.5 BioLegend) and CD8a (53-6.7 BioLegend) followed by staining with Streptavidin Pacific Orange (Invitrogen). Antibodies used to stain HSPCs included PE CD150 (TC15-12F123.2 BioLegend), PE Cy7 CD48 (HM48-1 BioLegend), PE Cy7 CD41 (MWReg eBiosciences), APC Sca1 (E13-161.7 BioLegend), APC Cy7 cKit (2B8 BioLegend). Antibodies used to stain peripheral blood included: FITC CD45.2 (104 BioLegend), PE CD3e (145-2C11 BioLegend), PE CD115 (AFS98 BioLegend), PE Cy5 B220 (RA3-6B2 eBioscience), PE Cy7 Gr-1 (RB6-8C5 BioLegend), PE Cy7 Ly6G (1A8 BD Pharmingen), APC Mac1 (M1/70 eBioscience), APC Cy7 CD45.1 (A20 eBioscience), biotin CD3e (145-2C11, eBioscience), biotin CD4 (GK1.5 BioLegend) and biotin CD8a (53-6.7 BioLegend).

Burberry A., Zeng M.Y., Ding L., Wicks I., Inohara N., Morrison S.J, & Núñez G. (2014). Infection Mobilizes Hematopoietic Stem Cells through Cooperative NOD-like Receptor and Toll-like Receptor Signaling. Cell host & microbe, 15(6), 779-791.

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