Gr 1 pecy7

Manufactured by Thermo Fisher Scientific

Sourced in Germany, China, United States

Gr-1-PECy7 is a fluorescent-labeled antibody used for the detection and quantification of Gr-1 (Ly-6G/Ly-6C) expressing cells in flow cytometry applications.

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12 protocols using gr 1 pecy7

Comprehensive Immune Cell Profiling

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Anti-mouse antibodies were purchased from BioLegend, eBioscience or Thermo Fisher for staining, including: CD3-BV510 (145-2C11), IL-7Rα-BV605 (A7R34), ICOS-PerCP.Cy5.5 (C398.4A), CD5-PE.Cy7 (53-7.3), CD8-PE.Cy7 (53-6.7), CD19-PE.Cy7 (eBio1D3), NK1.1-PE.Cy7 (PK136), CD11b-PE.Cy7 (M1/70), CD11c-PE.Cy7 (N418), Gr-1-PE.Cy7 (RB6-8C5), Fcϵ1-PE.Cy7 (MAR-1), TER-119-PE.Cy7 (TER-119), Siglec-F-Alexa Fluor 647 (E50-2440) and Live/Dead-A780. For flow cytometry, single-cell suspensions were generated and antibodies used at 1:200 for staining. Data were acquired using LSRFortessa flow cytometer and FACSDiva software, and analysed using FlowJo software.

Lok L.S., Walker J.A., Jolin H.E., Scanlon S.T., Ishii M., Fallon P.G., McKenzie A.N, & Clatworthy M.R. (2021). Group 2 Innate Lymphoid Cells Exhibit Tissue-Specific Dynamic Behaviour During Type 2 Immune Responses. Frontiers in Immunology, 12, 711907.

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UAMC-1110 Immunotherapy Protocol

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UAMC-1110 was generously provided by Dr. Pieter Van der Veken (University of Antwerp). Unless otherwise specified, UAMC-1110 was administered in high molecular weight PEG at a concentration of 20mg/kg by oral gavage twice per day. Fluorescently-conjugated antibodies CD3-e450, CD8-PerCP, CD4-FITC, CD4-e450, CD4-PerCP, CD25-APC, IFNγ-APC, IL-2-PE, TNFa-PE-Cy7, CD11b-PE-Cy7, Ly6G-FITC, Ly6C-PerCP-Cy5.5, Gr1-PE-Cy7, and MHCII-EF450, PDGFRβ-APC, CD31-PE, CD45-BV510, and EpCam-BV605 were purchased from Thermo Fisher Scientific (Lafayette, CO) or BD Biosciences (San Jose, CA). CD8-PE-TxRD was purchased from Invitrogen (Carlsbad, CA). SIY, SIINFEKL, and β-galactosidase peptides were obtained from Integrated DNA Technologies (Coralville, Iowa). β-galactosidase (βgal) for the ICPMYARV peptide and SIINFEKL peptide tetramers were obtained from the NIH Tetramer core facility (Atlanta, GA). Antibodies to CD31 (Thermo Fisher Scientific), αSMA (Sigma-Aldrich, St Louis, MO), PDGFRα (Thermo Fisher Scientific), Vimentin (Cell Signaling Technology, MA), Ki67 (Abcam), Cleaved Caspase 3 (Cell Signaling Technology) were used for immunofluorescent staining and hypoxyprobe Omni Kit (Burlington, MA) was used for hypoxia staining. Anti-PD1 antibody clone RPM1-14 (BioXcell, West Lebanon, NH) was used where indicated for treatment studies.

Gunderson A.J., Yamazaki T., McCarty K., Phillips M., Alice A., Bambina S., Zebertavage L., Friedman D., Cottam B., Newell P., Gough M.J., Crittenden M.R., Van der Veken P, & Young K.H. (2019). Blockade of fibroblast activation protein in combination with radiation treatment in murine models of pancreatic adenocarcinoma. PLoS ONE, 14(2), e0211117.

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Multiparametric Flow Cytometry of Immune Cells

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Flow cytometric assay was done as previously described.^{52 (link),53 (link)} Briefly, bone marrow cells isolated by flushing the tibias and femurs of the euthanized mice, resuspended in a single-cell suspension and undiluted whole blood cells were used for flow cytometric analysis. Following the removal of red blood cells, the remaining cells were stained with a cocktail of antibodies (CD11b (Mac-1)-PE, Gr-1-PECy7, CD68 APC, CD4-eFluoro450 (PB), B220APC, and CD8-APCCy7 from eBiosciences) to identify the population of myeloid, B cells, and T cells. Flow cytometry analysis was performed on a Becton Dickinson 5-laser LSRII instrument.

West J., Chen X., Yan L., Gladson S., Loyd J., Rizwan H, & Talati M. (2020). Adverse effects of BMPR2 suppression in macrophages in animal models of pulmonary hypertension. Pulmonary Circulation, 10(1), 2045894019856483.

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Flow Cytometry Analysis of Mouse Tumor Tissue

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Small samples of mouse tumor tissues (2–3 wk after last immunization) were chopped in 1 ml of cell dissociation solution (1% 100 mg/ml collagenase/dispase, 0.5% 20 mg/ml DNase in DMEM) and incubated for 30 min at 37°C. The cell suspension (1×10⁵ to 1×10⁶ cells) was stained for use in flow cytometry after blocking the Fc receptors by applying ultra-blocking buffer (10% normal mouse serum, 10% normal hamster serum, 10% normal rat serum, and monoclonal Ab 2.4G2 in PBS). Abs used were CD11c-FITC (eBioscience, San Diego, CA, USA), CD45-PE-Texas-Red (Invitrogen, Carlsbad, CA, USA), F4/80-PE-Cy5 (eBioscience), CD11b-biotin (Miltenyi, Bergisch Gladbach, Germany), Gr-1-PE-Cy7 (eBioscience), and SA-PerCP-Cy5.5 (eBioscience). All samples were filtered through a 200 μm nylon mesh before running in BD FACSAria. The results were analyzed by using the Flowjo 10 program (Tree Star, Inc., Ashland, OR, USA).

Kim K.Y., Cho E.H., Yoon M, & Kim M.G. (2020). Critical Adjuvant Influences on Preventive Anti-Metastasis Vaccine Using a Structural Epitope Derived from Membrane Type Protease PRSS14. Immune Network, 20(4), e33.

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Flow Cytometry Analysis of Parasitized Blood

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Freshly isolated ECs were stained in FACS buffer (0.5% BSA, 2 mM EDTA in PBS). Cells were stained with CD45-APC-eFluor-780 antibodies purchased from eBioscience and with CD31 (PECAM1)-AF488 and CD102(ICAM2)-AF647 purchased from Biolegend. The stained samples were run on an LSRFortessa cell analyser, and the results were analysed using FlowJo software (TreeStar). Whole blood was obtained by cardiac puncture. To prevent coagulation, whole blood was collected in 3.8% sodium citrate. For the detection of free parasites, whole blood was diluted in PBS and immediately subjected to analysis on the LSRFortessa cell analyser. Free parasites were identified by their size and expression of tdTomato. The gate for free parasite was drawn on freshly isolated tachyzoites from cell culture. Amnis ImageStream was used to visualize infected cells in the blood. White blood cells were purified using whole blood from infected mice, which was layered on top of Lympholyte (Cedarlane) and centrifuged at room temperature. The mononuclear preparation was removed and washed in FACS buffer. Cells were then stained in FACS buffer with CD11b-APC-e780, CD11c-APC and Gr1-PE-Cy7 from eBioscience and CD3-FITC from BD Biosciences. The stained samples were then used for analysis.

Konradt C., Ueno N., Christian D.A., Delong J.H., Pritchard G.H., Herz J., Bzik D.J., Koshy A.A., McGavern D.B., Lodoen M.B, & Hunter C.A. (2016). Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system. Nature microbiology, 1, 16001.

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Quantification of Inflammatory Cell Responses

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One day after the last exposure to PM, BALF cells were obtained with three 0.4 ml PBS washes injected into the lungs and then withdrawn to collect the cells. The total number of BALF cells were counted, and then the remaining BALF cells were centrifuged at 400 g for 10 min at 4 °C. The supernatant was stored at - 80 °C and used for analysis of cytokines. The cell pellet was suspended in 200 μl PBS and stained with Gr-1 (PE-Cy7, cat. 25-5931-81, eBioscience), CD11b (BV421, cat. 101236, Biolegend), Annexin V (MultiSciences, Hangzhou, China), and PI (MultiSciences, Hangzhou, China) for 30 minutes at 4 °C. Bcl-2 antibody was purchased from Santa Cruz Biotechnology (Dallas, US). Data were acquired with a FACScalibur flow cytometer and analyzed with FlowJo software (version 7.6, San Carlos, CA)

Geng X., Wang X., Luo M., Xing M., Wu Y., Li W., Chen Z., Shen H, & Ying S. (2018). Induction of neutrophil apoptosis by a Bcl-2 inhibitor reduces particulate matter-induced lung inflammation. Aging (Albany NY), 10(6), 1415-1423.

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Multiparameter Analysis of Bone Marrow Stromal Cells

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CD45^– BM stromal cells were stained using anti-mouse CD45-APC, CD45-FITC, CD45-PE, SCA-1-PE (all BD Biosciences), CD140a (PDGFRa)-PECy7, and CD274-APC (both Biolegend). Myeloid cells were identified using CD11b-FITC (Biolegend) or CD11b-PerCP-Cy5.5 (eBiosciences). MDSC were stained using CD11b-PerCP Cy5.5, Gr-1-PECy7 (eBiosciences), k and LyC-Alexa700 (Biolegend). For intracellular staining of CXCL12, cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X (PBS-T), and stained with SDF-1-Fluorescein (R&D; IC350F, 1:10). For surface staining, antibodies were used at 1:50 in PBS for 10min at room temperature and live cells negative for 4′,6-Diamidino-2-phenylindol (DAPI; Sigma: 1μg/ml) were analyzed on the Fortessa or sorted with the ARIA II cell sorter (both BD).

Cioffi M., Trabulo S.M., Vallespinos M., Raj D., Kheir T.B., Lin M.L., Begum J., Baker A.M., Amgheib A., Saif J., Perez M., Soriano J., Desco M., Gomez-Gaviro M.V., Cusso L., Megias D., Aicher A, & Heeschen C. (2017). The miR-25-93-106b cluster regulates tumor metastasis and immune evasion via modulation of CXCL12 and PD-L1. Oncotarget, 8(13), 21609-21625.

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Adoptive Transfer of Mpzl3-Deficient Lymphocytes

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Bone marrow was flushed out of femurs and tibias of Mpzl3 −/− or +/+ littermates (6 weeks old). The bone marrow was labeled using biotinylated antibodies against CD3, CD4, CD8 and B220 (eBioscience, San Diego, CA). Streptavidin-labeled microbeads were then used to magnetically deplete the biotinylated lymphocytes using autoMACS (Milteyni Biotec, Inc., Auburn, CA). 3 × 10⁶ bone marrow cells after depletion were injected intraperitoneally into 2-day old B6 Rag −/− mice (The Jackson Laboratory, Bar Harbor, ME). After 10 weeks, the inguinal lymph nodes and spleens of these mice were analyzed by flow cytometry, and skin analyzed by histology. For flow cytometry, 3 × 10⁶ cells were blocked using a cocktail of anti-CD16/32 (2.4G2) and normal mouse sera (Jackson ImmunoResearch, West Grove, PA) before being stained with fluorescently labeled antibodies to determine their phenotype and activation status. The following antibody conjugates were used: CD11b PE-TR (Life Technologies Corp.), CD4 V500 (Becton Dickinson, San Jose, CA), CD11c FITC, Gr-1 PECy7, CD3 Alexa Fluor 700, CD8 efluor605, B220 PerCpCy5.5, CD44 efluor450, CD62L APC, CD69 PE (eBioscience). Cells were then washed and re-suspended in 2% FBS in PBS for analysis with flow cytometers (LSR-II and Fortessa, Becton Dickinson).

Leiva A.G., Chen A.L., Devarajan P., Chen Z., Damanpour S., Hall J.A., Bianco A.C., Li J., Badiavas E.V., Zaias J., Miteva M., Romanelli P., Nouri K, & Wikramanayake T.C. (2014). Loss of Mpzl3 Function Causes Various Skin Abnormalities and Greatly Reduced Adipose Depots. The Journal of investigative dermatology, 134(7), 1817-1827.

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Characterizing Melittin Binding to Immune Cells

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Rhodamine-conjugated melittin peptides were purchased from GenScript (Piscataway, NJ, USA). Splenocytes were seeded in a 6-well culture plate with 0.5 μg/ml of rhodamine-conjugated melittin. An hour later, cells were harvested and unbound peptides were washed off twice. Cells were stained with APC-conjugated antibodies for an hour at 4 °C to confirm the binding of melittin to CD4⁺ and CD8⁺ T cells and CD11b⁺ monocytes.
To verify whether the binding of melittin to CD11b⁺ cells is related to phagocytosis, splenocytes were pretreated with 10 nM cytochalasin D or vehicle (DMSO) for an hour in a 37 °C incubator. Next, the cells were incubated with the rhodamine-conjugated peptides and stained with CD11b-APC antibody as described above.
To observe the binding of melittin on CD11b⁺ subsets in splenocytes, macrophage, dendritic cells, and neutrophils were probed with anti-mouse F4/80-FITC, CD11c-APCcy7, and Gr1-PEcy7 (e-bioscience). Annexin-V was added to the samples prior to data acquisition to discriminate dead cells. M1 of M2 macrophages were stained with CD86-PEcy7 (e-bioscience) or CD206-PercpCy5.5 (Biolegend). Cells were detected on a FACSCalibur or FACSCantoII.

Lee C., Bae S.J., Joo H, & Bae H. (2017). Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model. Oncotarget, 8(33), 54951-54965.

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Flow Cytometric Analysis of BAL Fluid Cells

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The BAL fluid cell suspensions were obtained from either mice or in vitor cell culture medium (RPMI 1640 with 10% FBS, Thermo Fisher Scientific) at specific time points. The expression of cell surface markers was assessed by incubating the samples with fluorescent dye-conjugated mouse antibodies against Gr-1 (Pe-Cy7) and SiglecF (PE) (eBioscience, San Diego, CA, USA) for 30 min at 4 °C. Based on the surface marker staining, the suspensions were incubated with Annexin V (FITC) and propidium iodide (PI) (MultiSciences, Hangzhou, Zhejiang, China) to assess BAL fluid cell apoptosis. The data were acquired using a FACSCalibur flow cytometer (FC500) (BD Biosciences, Sparks, MD, USA) and analyzed using FlowJo software ver. 7.6 (Tree Star, San Carlos, CA, USA).

Tian B.P., Li F., Li R., Hu X., Lai T.W., Lu J., Zhao Y., Du Y., Liang Z., Zhu C., Shao W., Li W., Chen Z.H., Sun X., Chen X., Ying S., Ling D, & Shen H. (2018). Nanoformulated ABT-199 to effectively target Bcl-2 at mitochondrial membrane alleviates airway inflammation by inducing apoptosis. Biomaterials, 192, 429-439.

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