Cells were harvested from the BM, SP, and FL of a WT mouse, stained in FACS buffer with FVD780 (eBioscience), F4/80-AF647, CD169-PE, and VCAM-AF488 (Biolegend), and analyzed on a FACSCanto flow cytometer (BD). The data were analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA).
Fvd780
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FVD780 is a laboratory instrument designed for precise liquid handling and dispensing. It features a compact and robust design to ensure reliable performance in a variety of laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto, by BD (1 mentions)
F4 80 af647, by BioLegend (1 mentions)
Cd169 pe, by BioLegend (1 mentions)
Fc block, by BD (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Human fc block, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Ifn γ apc, by BD (1 mentions)
Facs staining buffer, by BD (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Pd l1 apc, by BD (1 mentions)
6 protocols using fvd780
1
Immunophenotyping of Myeloid Cells
2
Lung and BAL Immune Cell Analysis
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Lung and BAL samples were incubated with live/dead staining dye FVD780 (eBioscience) followed by washing with PBS. Then, samples were fixed with 2% paraformaldehyde. After washing, the samples were blocked with Fc block (BD Biosciences) for 10 min on ice. Next, cell suspensions were stained for the surface Ags Ly6G FITC, Ly6c v450, NK1.1 allophycocyanin, CD11c PE-Cy7, CD11b PE-CF594, F4-80 PE, and MHCII Alexa Fluor 700. Respective isotype Abs (Iso ham PE-Cy7, IgG2b PE, IgG2a allophycocyanin, IgM v450) were used to set negative gates. Samples were run on a Gallios flow cytometer, and analysis was performed with Kaluza software (Supplemental Fig. 2 ).
3
Quantifying Autophagy Inhibition in Huh7 Cells
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Huh7 cells infected at greater than 90% were transfected with either mock (pcDNA3-mRuby2) or ATG4B-DN. At day 2 cells were trypsinized and washed twice with PBS. Dead cells were labeled using FVD780 (eBioscience). Cells were fixed with 4% formaldehyde for 10 min at RT, incubated with blocking buffer (PBS, 2% BSA, 0.2% Saponin) for 20 min at 4°C and incubated with primary antibodies diluted in blocking buffer for 30 min at 4°C. After washing with PBS, cells were incubated with Alexa fluor-488 goat anti-rabbit IgG (Invitrogen) for 30 min at 4°C. Cells were analyzed using BD LSRFortessa cell analyzer (BD Biosciences) and Cyflogic software (CyFlo Ltd).
4
Phenotypic Analysis of CD161+ CD4+ T Cells
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Peripheral blood mononuclear cells and LILs were prepared for phenotypic analysis of circulating and intrahepatic CD161+CD4+ T cells. Human Fc Block™ (BD Bioscience, USA) was used to block non‐specific binding. Dead cells were excluded using a fixable viability dye (FVD)‐780 (eBioscience). Negative controls were set appropriately. FMO (fluorescence minus one) staining was performed to set a proper gate for indicators without an obvious bimodal distribution of expression. For staining of intracellular cytokines, cells were pre‐treated with a cell stimulation cocktail containing phorbol‐12‐myristate‐13‐acetate (PMA) and ionomycin (2 μL mL‐1) (eBioscience) for 5 h. A minimum of 1 × 105 cells were collected by LSRFortessaTM (BD Bioscience) for further analysis using FlowJo V10 software (Tree Star).
5
Comprehensive Immune Profiling of Brain Cells
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Brains were excised after euthanasia and single-cell suspensions were obtained by mincing brain and digestion with a gentleMACS Dissociator and a Tumor Dissociation Kit, human (Miltenyi Biotec, Bergisch Gladbach, Germany). Single-cell suspensions were incubated with anti-Fcγ receptor antibody (Clone 2.4G2, Tonbo Biosciences, San Diego, CA, USA) and the fixable viability dye FVD780 (Thermo Fisher Scientific), then stained with the following monoclonal antibodies; mouse CD45 (30-F11), CD3 (17A2), CD49b (DX5), CD8 (KT15), CD4 (GK1.5), Foxp3 (FJK-16s), granzyme B (GB11), CD69 (H1.2F3), Ki67 (B56), PD-1(29F.1A12), human EGFR (AY13), human PD-L1 (MIH1 or 29E.2A3) from BD Biosciences (San Jose, CA, USA), BioLegend (San Diego, CA, USA), Thermo Fisher Scientific, or Medical & Biological Laboratories (Nagoya, Japan). The appropriate conjugated isotype-matched IgG was used as the control if necessary. Intracellular staining was performed using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Stained cells were analyzed using LSRFortessa X-20 cell analyzer (BD Biosciences) and data analyzed with FlowJo 10 software (Tree Star, San Carlos, CA, USA). The gating strategies were shown in Supplementary Figs. S1 and S2.
6
Multiparameter Flow Cytometry for Immune Profiling
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The following antibodies were used: antihuman CD3-FITC or V500, CD4 PerCP-Cy5.5, CD8 FITC or V450, CD56 PE or BV510, IFN-γ APC, PD-L1 APC, and mIgG1 APC isotype control (all from BD Biosciences). Cell surface staining was performed by incubating cells with mAbs for 30 min at 4°C in FACS staining buffer (BD Biosciences) followed by 2 times washing with phosphate-buffered saline. For intracellular staining, surface stained cells were fixed and permeabilized using fixation/permeabilization buffer (Thermo Fisher Scientific) and stained with anti-IFN-γ mAb according to the manufacturer’s instructions. Samples were resuspended in FACS buffer and acquired using a LSRFortessa flow cytometer (BD Biosciences) with FACSDiva software and analyzed using FlowJo software (FlowJo). For FACS data analysis, doublets were excluded using forward scatter height and width properties and dead cells were excluded by FVD780 (Thermo Fisher Scientific) positive staining.
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