The mice were euthanized at 4 weeks after MI. Their blood, spleens, bone marrow and infarcted hearts were collected and made into single-cell suspensions for flow cytometry analysis, as previously described10 (link). The cells were stained with a mixture of antibodies (anti-CD11b-APC and anti-Gr-1-PerCP-Cy5.5 BD Biosciences). The data were acquired using an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo7 software (Tree Star, Inc.).
Anti gr 1 percp cy5
Manufactured by BD
Sourced in United States
Anti-Gr-1-PerCP-Cy5.5 is a fluorescent-labeled antibody used for the detection and identification of Gr-1 positive cells in flow cytometry applications. It is conjugated with PerCP-Cy5.5, a tandem dye that combines the properties of peridinin chlorophyll protein (PerCP) and cyanine 5.5 (Cy5.5) for multicolor analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b apc, by BD (4 mentions)
Flowjo7, by Tree Star (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti ly6c pe, by BD (1 mentions)
Serum free medium, by Thermo Fisher Scientific (1 mentions)
Anti ter119 apc, by BD (1 mentions)
Anti cd41 pe, by BD (1 mentions)
Prism v6, by GraphPad (1 mentions)
Facsarray, by BD (1 mentions)
Il 33, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Fc block, by BD (1 mentions)
Anti ly6g clone 1a8, by BioLegend (1 mentions)
Anti gr 1 clone rb6 8c5, by BioLegend (1 mentions)
Anti ly6g pe cy7, by BD (1 mentions)
Anti cd45 apc cy7, by BD (1 mentions)
Rat igg2a, by BioLegend (1 mentions)
Anti cd11b fitc, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Anti cd45 fitc, by BD (1 mentions)
6 protocols using anti gr 1 percp cy5
1
Quantifying post-MI immune cells
2
Analyzing Immune Cells Post-Myocardial Infarction
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Mice were sacrificed at 1 day and 7 days after MI. The blood, spleen, bone marrow and infarcted myocardium were collected and made into single-cell suspensions for flow cytometry, as previously described26 (link)52 (link). The cells were stained with a mixture of antibodies (anti-CD11b-APC, anti-Gr-1-PerCP-Cy5.5, anti-Ly6C-PE; BD Biosciences). Data were acquired using an LSRII flow cytometer (BD Biosciences) and were analyzed with FlowJo7 software (Tree Star, Inc.).
3
Single-cell multilineage differentiation assay
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Single CD34-LKS cells from C57BL/6 mice were individually deposited into each well of a 96-well plate along with 100 μL of serum free medium (Gibco) containing 100 ng/mL of mouse SCF, 10 ng/mL of mouse IL-3, 25 ng/mL of human TPO, 2 U/mL of human EPO and p18SMI compounds (different dilutions). Cells were cultured in a 37 °C, 5% CO2 incubator for 14 days for ex vivo colony formation. Colonies were then cytospined and analyzed using flow cytometry. Neutrophils (n), macrophages (m), erythroblasts (E) or megakaryocytes (M) were identified based on their cell surface maker, Gr-1, Mac-1, Ter119 and CD41. Cells were measured on an FACS analyzer (FACSArray, BD Biosciences). Antibodies were used as following: anti-Gr-1-Percp-cy5.5, anti-Mac-1-APC-cy7, anti-Ter119-APC, anti-CD41-PE (BD Biosciences). The selection of cell surface marker of each subpopulation cells were considered based on the manufacture’s guide (BD Biosciences). To be specific, Gr-1 cell marker represents neutrophils (n), Mac-1 represents macrophages (m), Ter119 represents erythroblasts (E) and CD41 represents megakaryocytes (M). The wells from each group that generated all lineages (nmEM) were counted. The ratio of p18SMI compound treatment group to the control group (DMSO-treated) was calculated and fitted to a curve to calculate the ED50 value using GraphPad Prism v6.0.
4
Neutrophil Profiling in Inflammatory Stimuli
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Mice were injected intraperitoneally with 100 μL PBS, or 5 μg kg−1 IL‐33 (PeproTech) or 2.5 mg kg−1 LPS (Escherichia coli strain O26:B6; Sigma; L2654) in 100 μL PBS. After 3 h, the mice were sacrificed via an increasing concentration of CO2, and the peritoneal cavity was washed using 4 mL PBS containing 1% BSA and 5 mm EDTA. Cells were pelleted by centrifugation and incubated on ice with Fc Block (1 in 50 dilution, BD Biosciences) for 10 min. Cells were then stained with anti‐Gr1‐PerCp‐Cy5.5 (1 in 800, BD Biosciences, #552093) and anti‐CD11b‐APC (1 in 1600, BD Biosciences #553312) for 30 min. Cells were then washed twice and analyzed by flow cytometry. For analysis, live cells were gated based on a forward and side scatter and neutrophils defined as CD11b Gr1 double‐positive cells. Gating and representative FACS plots are shown in Supplementary figure 12 .
5
Neutrophil Depletion in Murine CVB3 Model
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To deplete neutrophils in mice, 250 μg (12.5 mg/Kg) of anti-Ly-6G (clone 1A8, Biolegend) and isotype control (rat IgG2a, Biolegend) Abs, or 200 μg (10 mg/Kg) of Anti-Gr-1 (clone RB6-8C5, Biolegend) in 100 μl PBS were injected i.p. into mice 24 h prior to and 3 days after inoculation with CVB3 (Day 0). To evaluate influence of neutrophils on the development of cardiac fibrosis, mice received Abs injection on days −1, 3, 6, 9, 12, 15, and 18. Blood was collected by saphenous venous puncture. Total white blood cell counts (WBC) were determined by visual enumeration after trypan blue exclusion. The percentage of neutrophils was determined by flow cytometry using antibody cocktails (anti-CD45-APC/Cy7, anti-CD11b-FITC, anti-Gr-1-PercP/Cy5.5, and anti-Ly6G-PE/Cy7, BD PharMingen). Fluorescent intensity was determined using a FACS CantoII flow cytometer (BD Bioscience) and the data were analyzed using FlowJo v10.0 software (Tree Star).
6
Mouse Model for Liver Inflammation
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Eighty male C3H mice (6 weeks old, weighing 20–25 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, china), and kept in a specific pathogen free mouse breeding room with controlled temperature of 25 ± 1°C. The mice were provided free access to food.
Anti-CD45 FITC, anti-F4/80 PE, anti-CD11b APC, and anti-Gr1PerCP-Cy5.5 were purchased from BD Biosciences (Lake Franklin, New Jersey, United States). Anti-CD3 FITC antibody was purchased from BioLegend (San Diego, California, United States). Primary antibodies against WNT1 (H-89) and NF-KB P65(F-6) were purchased from Santa Cruz Biotechnology Co., Ltd. (Santa Cruz Avenue, California, United States). Primary antibodies against VEGFA (VG-1), β-catenin, and APC were purchased from Abcam (Cambridge, MA, United States). Primary antibody against β-actin was purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Anti-CD31 was purchased from Abcam. DEN was purchased from Merck Group (Darmstadt, Germany).
Anti-CD45 FITC, anti-F4/80 PE, anti-CD11b APC, and anti-Gr1PerCP-Cy5.5 were purchased from BD Biosciences (Lake Franklin, New Jersey, United States). Anti-CD3 FITC antibody was purchased from BioLegend (San Diego, California, United States). Primary antibodies against WNT1 (H-89) and NF-KB P65(F-6) were purchased from Santa Cruz Biotechnology Co., Ltd. (Santa Cruz Avenue, California, United States). Primary antibodies against VEGFA (VG-1), β-catenin, and APC were purchased from Abcam (Cambridge, MA, United States). Primary antibody against β-actin was purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Anti-CD31 was purchased from Abcam. DEN was purchased from Merck Group (Darmstadt, Germany).
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