Kidneys were minced into ~1 mm3 pieces and then digested with 0.5 mg/mL Collagenase D (Sigma), 0.5 mg/mL DNAse (Sigma) and 1 mg/mL Dispase (Stemcell Technologies) for 45 minutes at 37°C (200 rpm). Thereafter, the solution was filtered through 100 μm mesh, and the supernatant pelleted (1600 rpm, 5 minutes at 4°C), enriched through a percoll gradient (40 to 80%), and kidney leukocytes were collected and analyzed by FACS. Blood obtained from the retro-orbital sinus was immediately placed in heparinized tubes and washed with PBS followed by RBC lysis buffer (10 mM KHCO3, 16 mM NH4Cl, pH 7.3) prior to FACS analysis. Fluorophore-conjugated antibodies used for flow cytometry include B220 (RA3-6B2, BioLegend), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD11b (M1/70, BioLegend), CD45 (30-F11, eBioscience), Foxp3 (FJK-16S, eBioscience), IL-17A (eBio17B7, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8, BD Biosciences), Ly6G (RB6-8C5, eBioscience), RORγT (AFKJS-9, eBioscience). For neutrophil depletion, 500 μg of purified anti-Ly6G (1A8, BioXcell) or isotype (Rat IgG2a, BioXcell) antibody was administered by intraperitoneal injection 10 days after C. albicans infection with sustained dosing every three days thereafter.
Ly6g rb6 8c5
Manufactured by Thermo Fisher Scientific
Ly6G (RB6-8C5) is a monoclonal antibody that recognizes the Ly6G antigen, which is specifically expressed on neutrophils. This antibody can be used for the identification and isolation of neutrophils in flow cytometry and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd8 53 6, by Thermo Fisher Scientific (3 mentions)
Ly6c hk1, by Thermo Fisher Scientific (3 mentions)
Cd4 gk1, by Thermo Fisher Scientific (3 mentions)
Dnase, by Merck Group (2 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Ebio17b7, by Thermo Fisher Scientific (2 mentions)
Ly6g 1a8, by BD (2 mentions)
Rorγt afkjs 9, by Thermo Fisher Scientific (2 mentions)
Cd45 30 f11, by Thermo Fisher Scientific (2 mentions)
B220 ra3 6b2, by BioLegend (2 mentions)
Cd11b m1 70, by BioLegend (2 mentions)
Anti ly6g 1a8, by BioXCell (2 mentions)
Rat igg2a, by BioXCell (2 mentions)
Dispase, by STEMCELL (2 mentions)
Facsaria, by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (1 mentions)
4 protocols using ly6g rb6 8c5
1
Kidney Leukocyte Isolation and Neutrophil Depletion
2
Kidney Leukocyte Isolation and Neutrophil Depletion
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Kidneys were minced into ~1 mm3 pieces and then digested with 0.5 mg/mL Collagenase D (Sigma), 0.5 mg/mL DNAse (Sigma) and 1 mg/mL Dispase (Stemcell Technologies) for 45 minutes at 37°C (200 rpm). Thereafter, the solution was filtered through 100 μm mesh, and the supernatant pelleted (1600 rpm, 5 minutes at 4°C), enriched through a percoll gradient (40 to 80%), and kidney leukocytes were collected and analyzed by FACS. Blood obtained from the retro-orbital sinus was immediately placed in heparinized tubes and washed with PBS followed by RBC lysis buffer (10 mM KHCO3, 16 mM NH4Cl, pH 7.3) prior to FACS analysis. Fluorophore-conjugated antibodies used for flow cytometry include B220 (RA3-6B2, BioLegend), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD11b (M1/70, BioLegend), CD45 (30-F11, eBioscience), Foxp3 (FJK-16S, eBioscience), IL-17A (eBio17B7, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (1A8, BD Biosciences), Ly6G (RB6-8C5, eBioscience), RORγT (AFKJS-9, eBioscience). For neutrophil depletion, 500 μg of purified anti-Ly6G (1A8, BioXcell) or isotype (Rat IgG2a, BioXcell) antibody was administered by intraperitoneal injection 10 days after C. albicans infection with sustained dosing every three days thereafter.
3
Multiparameter Flow Cytometry Immunophenotyping
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The cells were processed as described in the tissue preparation section of the methods. After processing, the cells suspensions were blocked with a CD16/32 antibody for 10 min at 4 °C and stained with fluorochrome labelled antibodies at 4 °C for 30 min. After the staining was performed, the cell were rinsed with PBS containing 0.2% BSA for analysis. For intracellular antigen staining, the Foxp3/Transcription Factor Staining Buffer Set (00-5523, eBioscience) was used according to the manufacturer’s protocol after the surface staining was complete. Peridinin chlorophyll protein (PerCP) Cyanine5.5-conjugated anti-CD45 (30-F11), phycoerythrin (PE)-conjugated anti-CD3e (145-2C11), fluorescein isothiocyanate (FITC)-conjugated anti-γδTCR (GL3), allophycocyanin (APC)-conjugated anti-IL-17A (eBio17B7), and Ly-6G (RB6-8C5) were purchased from eBiosciences. The samples were analysed using a FACScalibur or were sorted using a FACSAria (BD Biosciences, Franklin Lakes, NJ, USA). The samples were analysed using FlowJo software 10.6 (Tree Star, Inc.).
4
Multiparameter Flow Cytometry of Murine and Human Myeloid Cells
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Murine cells were stained using standard protocols for the surface markers CD45 (clone 30-F11), CD11b (M1/70, eBioscience), Ly6C (HK1.4, eBioscience), Ly6G (RB6-8C5, eBioscience), F4-80, (BM8, eBioscience), MHCII (M5/114.15.2, BD), CD86 (GL1, eBioscience), CD4 (GK1.5, eBioscience), CD8 (53-6.7, eBioscience), CD25 (PC61.5, eBioscience), and LIVE/DEAD Fixable Near-IR Dead Cell Stain. Intracellular staining for Arginase 1 (R&D) (polyclonal sheep IgG), iNOS (CXNFT, eBioscience), TNFa (MP6-XT22, eBioscience) was performed on cells fixed/permeabilized with the Transcription Factor Staining Buffer Set (eBioscience). Human MDSCs were stained for the surface markers CD11b (ICRF44, eBioscience), CD33 (WM-53, eBioscience), HLA-DR (L234, BioLegend), CD4 (OKT4, eBioscience), CD8 (HIT8A, eBioscience). Cytokine production was assessed after 3 hours of PMA + Ionomycin stimulation. Cells were analyzed on the BD Symphony and sorted on the BD FACS Aria.
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