Approximately 106 cells were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA) and with a Zombie Aqua™ viability dye (BioLegend, San Diego, CA, USA). After washing with PBS + 2% fetal calf serum (FCS), the cells were stained for 30 min with the fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (cat. No. 11-0031-81, eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-Ly6G (cat. No. 12-9668-82, eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-F4/80 (cat. No. 17-4801-80, eBioscience, San Diego, CA, USA) and PERCP-Cy5.5-conjugated anti-CD11b (cat. No. 45-0112-80, eBioscience, San Diego, CA, USA). Next, cells were washed twice and fixed with 0.37% formaldehyde in PBS. Cells were analyzed on a BD LSR Fortessa X20 with DIVA software (BD, Franklin Lakes, NJ, USA, v9.0). Results were further analyzed with the FlowJo software (BD, Franklin Lakes, NJ, USA, v10.0). Flow cytometry plots showing the gating strategy for cellular identification are depicted in Supplementary Figure S2 .
Phycoerythrin pe conjugated anti ly6g
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phycoerythrin (PE)-conjugated anti-Ly6G is a fluorescently labeled antibody that binds to the Ly6G antigen. It is used for the detection and analysis of Ly6G-expressing cells in flow cytometry applications.
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Lab products found in correlation
Zombie aqua viability dye, by BioLegend (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Xylazine, by Syntec (2 mentions)
Ketamine, by Syntec (2 mentions)
Sytox green, by Thermo Fisher Scientific (2 mentions)
Percp cy5.5 conjugated anti cd11b, by Thermo Fisher Scientific (1 mentions)
Diva software, by BD (1 mentions)
Pe cy5 conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Liberase tl enzyme cocktail, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Liberase tl, by Roche (1 mentions)
Buv395 conjugated anti cd45, by BD (1 mentions)
Eclipse ti, by Nikon (1 mentions)
4 protocols using phycoerythrin pe conjugated anti ly6g
1
Multiparametric Flow Cytometry Analysis of Immune Cell Populations
2
In vivo Imaging of Liver Leukocytes
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In vivo imaging was performed as previously described [12 (link)]. Briefly, mice were anesthetized intraperitoneally (i.p.) with a ketamine and xylazine solution (Syntec, São Paulo, Brazil, 60 mg/kg and 15 mg/kg, respectively) and a midline laparoscopy was performed to expose the liver for imaging. Prior to surgery, mice were injected, intravenously (i.v.), with Phycoerythrin (PE)-conjugated anti-Ly6G (4 µg, eBioscience, San Diego, CA, USA clone 1A8) and Sytox Green (5 nmol/mouse of Invitrogen). Labeled antibodies and florescent probes were diluted in a total volume of 100 µL before injection. Mice were imaged using a Nikon Eclipse Ti with an A1R confocal microscope loaded with a spectral detector and XYZ motorized stage.
3
Wound Tissue Single-Cell Isolation
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Following humane euthanasia, wounds were harvested using 6 mm punch biopsy to collect 2 mm wound margins for analysis. Three pools of 2 wounds per animal were chopped and digested with Liberase TL enzyme cocktail [0.35 mg/mL Liberase TL (Roche), 3 mg/mL Collagenase D (Roche) and 0.1 mg/mL DNase I (Roche)] for 2 h at 37°C.25 (link) After incubation, the samples were filtered through a 70-µm strainer to remove undigested debris and to yield single-cell suspensions. Approximately 1×106 cells were incubated with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences) and with a Zombie Aqua™ viability dye (BioLegend) for 15 min. After washing with FACS buffer (0.5% bovine serum albumin (BSA); 2 mM EDTA in PBS), surface receptors were stained with BUV395-conjugated anti-CD45 (BD), BV786-conjugated anti-CD11b (BD), phycoerythrin (PE)-conjugated anti-Ly6G (eBioscience) and PE/Cy5-conjugated anti-F4/80 (eBioscience) for 30 min. Next, cells were washed and fixed with 0.37% formaldehyde in PBS. Cells were analyzed on a BD LSR Fortessa X20 with DIVA software. Results were further analyzed with the FlowJo software (BD Biosciences).
4
Intravital Imaging of Liver Inflammation
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Confocal intravital imaging was performed as previously described [3 (link)]. Briefly, mice were anesthetized (i.p.) with a mixture of ketamine and xylazine (Syntec, 60 mg/kg and 15 mg/kg, respectively) and a midline laparoscopy was performed to expose the liver for imaging. Prior to surgery, mice were injected i.v. with 1 µL of Sytox Green, (Invitrogen, Carlsbad, CA, USA) and Phycoerythrin (PE)-conjugated anti-Ly6G (4 µg, eBiosciences, San Diego, CA, USA clone 1A8). Positive labeling was confirmed by injection of matched isotype control (PE-rat anti-mouse IgG). Labeled antibodies and fluorescent probes were diluted in a total volume of 100 µL before injection. Mice were imaged using a Nikon Eclipse Ti (Nikon, Shinagawa, Tokyo, Japan) with an A1R confocal microscope loaded with a spectral detector and XYZ motorized stage. To confirm type 1 IFN production in our Yellow Fluorescent Protein (YFP)-expressing strain, mice were treated with R848—resiquimod (TLR7 and TLR8 agonist); 50 nmol/mouse, i.p., 24 h before imaging.
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