Peritoneal cells were collected as above described. And the collected PLF was filtered to remove debris through a 100-μM filter. Then, neutrophils were isolated by using the Neutrophil Isolation Kit (Miltenyi Biotec). Briefly, resuspended cells were incubated with a neutrophil biotin-antibody cocktail for 10 minutes in the refrigerator (2-8°C), then anti-biotin microbeads were added. After incubating for 15 minutes, the cell suspension was applied to MS column, and flow-through cells were collected. The purity of the collected neutrophils was greater than 90%, as confirmed by using flow cytometry staining with CD11b and Ly6-G antibodies. For in vitro phagocytosis assays, isolated neutrophils were incubated with 1×106 CFU of fluorescently labeled E. coli (Abcam) in 24-well culture plates. After 2-hours incubated at 37°C in 5% CO2, cells were harvested and washed with PBS to remove unphagocytosed bacteria. For bacterial phagocytosis assay, the FITC-labeled Escherichia coli was measured by flow cytometry. FACS analysis was performed using a BD Accuir C6 Plus cell analyzer (BD). All data were analyzed using FlowJo (Tree Star). For all samples, at least 5×104 cells were collected to generate scatter plots.
Accuir c6 plus cell analyzer
Manufactured by BD
The BD Accuri C6 Plus cell analyzer is a compact, benchtop flow cytometer designed for a wide range of cell analysis applications. It features a blue laser and red laser for simultaneous detection of up to 6 fluorescent parameters, along with forward and side scatter measurements. The instrument is capable of analyzing up to 35,000 events per second with a sample volume as low as 12 microliters.
Automatically generated - may contain errors
Lab products found in correlation
Ros brite, by AAT Bioquest (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Percp conjugated rat igg2b, by BioLegend (1 mentions)
Apc conjugated rat igg1, by BioLegend (1 mentions)
Fitc conjugated rat igg2b, by BioLegend (1 mentions)
Pe conjugated rat igg2a, by BioLegend (1 mentions)
Fitc anti cd11b m1 70, by BioLegend (1 mentions)
Cxnft, by Thermo Fisher Scientific (1 mentions)
4 protocols using accuir c6 plus cell analyzer
1
Neutrophil Phagocytosis of E. coli
2
Neutrophil Identification via ROS-Brite Staining
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Cells from peritoneal lavage were plated at 5-10 ×105 cells in 96 well plates and incubated in complete media containing 5 μM/ml ROS Brite™ (10 µM, AAT Bioquest) for 30 minutes at 37 °C. The dye-loading solution was replaced with an HHBS buffer. And then the cells were washed with FACS buffer prior to being stained with the surface markers CD11b and Ly6G to identify neutrophils. FACS analysis was performed using a BD Accuir C6 Plus cell analyzer (BD). All data were analyzed using FlowJo (Tree Star).
3
Multicolor Flow Cytometry to Analyze Immune Cells
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Cells present in the PLF or obtained from omentum were pretreated on ice for 20 min with anti-mouse CD16/32 abs to block FC receptors. For cell surface staining, cells were stained on ice for 30 min with the following fluorescently conjugated antibodies: peridinin-Chlorophyll-Protein Complex (PerCP)-anti-CD45 (30-F11), phycoerythrin (PE)-anti-CD11b (M1/70), isothiocyanate (FITC)–anti-Ly6G (1A8), PE-anti-CD19 (6D5), and FITC–anti-CD11b(M1/70) (all from BioLegend). Neutrophils were identified by CD11b+Ly6G+, and B1 cells were identified by CD19+CD11b+. For intracellular staining, a cytofix/Cytoperm kit (BD) was used to fixed and permeabilized cells. Allophycocyanin (APC)–anti-TNF-α (MP6-XT22) and APC–anti-IL-6 (MP5-20F3) Ab were used for intracellular staining. Isotype controls used were PerCP-conjugated rat IgG2b, PE-conjugated rat IgG2a, FITC-conjugated rat IgG2b, and APC-conjugated rat IgG1 (all from BioLegend). FACS analysis was performed using a BD Accuir C6 Plus cell analyzer (BD). All data were analyzed using FlowJo (Tree Star). For all samples, at least 5×104 cells were collected to generate scatter plots.
4
Phenotypic Characterization of Innate Immune Cells
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Cells were blocked for Fc receptors with anti-mouse CD16/32 antibodies. For surface staining, cells were stained on ice for 30 mins with the following fluorescent conjugated antibodies: fluorescein isothiocyanate (FITC)–anti-Ly6G (1A8, Biolegend), peridinin-Chlorophyll-Protein Complex (PerCP)-anti-Ly6C Abs (HK1.4, Biolegend), phycoerythrin (PE)-anti-CD11b (M1/70, Biolegend) or allophycocyanin (APC)–anti-CD11b (M1/70, Biolegend). Staining for intracellular iNOS with APC–anti-iNOS (CXNFT, ThermoFisher scientific) was performed after fixation and permeabilization of the cells with a Cytofix/cytoperm kit (BD). Neutrophils were identified as CD11b+Ly6G+, and monocytes were identified as CD11b+Ly6G-Ly6C+ (Supplementary Figures 1 4
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