The IL-10RB−/− and control Mφs were grown in tissue culture plastic dishes in RPMI medium. Cells were detached using lidocaine-EDTA solution as described previously (Mukhopadhyay et al., 2006 (link)). Mφs were transferred into 96-well round-bottom plates at a density of 105 cells/well and incubated for 30 min at 4°C in 100 µl of FACS blocking buffer containing 5% FCS in PBS, 0.1% sodium azide, and 2 µl of Trustain Fc receptor block. After incubation, 5 µl of directly conjugated anti-human antibodies against individual Mφ plasma membrane antigens CD14 A488, CD16 APC-Cy7 (AbD Serotec), and CD206 APC (Becton Dickinson) or appropriate isotype-matched control antibodies with the same fluorophore were added to each well and incubated for an additional 30 min. Cells were washed twice with FACs buffer, resuspended in PBS, and analyzed on a Becton Dickinson FACsAria11 using FACS Diva software. For analysis of intracellular phosphorylated STAT3, cells were cultured for 15 min with 20 ng/ml rhIL-10 fixed with BD cytofix and permeabilized with ice-cold BD perm buffer III stained with anti-pSTAT3 (pY705)-Alexa Fluor 647 (clone 4/P-STAT3) according to the manufacturer’s Phosflow protocol (BD Biosciences). Signals were acquired on a BD LSR Fortessa (BD Biosciences) with FACS-Diva software (BD Biosciences). Data were analyzed using Flowjo v10.1 software.
Phosflow protocol
Manufactured by BD
Sourced in United States
The BD Phosflow Protocol is a laboratory equipment product designed to detect and analyze the phosphorylation status of cellular proteins. It provides a standardized workflow for sample preparation, staining, and flow cytometric analysis of phosphorylated intracellular targets.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cd206 apc, by BD (1 mentions)
Cd16 apc cy7, by Bio-Rad (1 mentions)
Facsaria11, by BD (1 mentions)
Perm buffer 3, by BD (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Cytofix, by BD (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Bioanalyzer 6000 pico chip, by Agilent Technologies (1 mentions)
Fc receptor blocking reagent, by Miltenyi Biotec (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Cd19 6d5, by BioLegend (1 mentions)
Ly6c hk1, by BioLegend (1 mentions)
Cd3 145 2c11, by BioLegend (1 mentions)
Cd69 h1.2f3, by BioLegend (1 mentions)
Cd86 gl 1, by BioLegend (1 mentions)
Cd20 sa275a11, by BioLegend (1 mentions)
Cd45r b220 ra3 6b2, by BioLegend (1 mentions)
7 protocols using phosflow protocol
1
Characterization of IL-10 Receptor Knockout Macrophages
2
Phosphorylation Analysis of Signaling Markers
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For examining phosphorylation of signalling markers with K562 stimulation, PBMC co-cultures plus IL-2 were established as described above. One day pi PBMCs were collected and either left unstimulated, or combined with K562 target cells at a 1:2 ratio (K562 to PBMC) for 2, 5, 10 or 30 mins on a rocking platform at 37°C. Following the BD Biosciences Phosflow protocol, activation was terminated by immediately fixing with 1 ml warmed 4% formaldehyde (Cytofix; BD Biosciences) for 10 mins at 37°C. Cells were then washed with FACS buffer and permeabilised by slowly adding 1 ml Perm Buffer III (pre-cooled to -20°C) (BD Biosciences) while vortexing, then incubated on ice for 30 mins. Cells were subsequently washed before resuspension in FACS buffer with antibodies at room temperature for 60 mins. Finally, cells were washed again and suspended in FACS buffer for acquisition on an LSR-II cytometer (BD Biosciences).
3
Comprehensive Immune Cell Profiling Protocol
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PBMCs (0.3x10 6 ) were incubated with Fc-receptor blocking reagent (Miltenyi Biotec) followed by staining for surface markers (Supplementary Table S2). Intracellular staining was performed using the Cytofix/Cytoperm kit (BD Biosciences). Phosphorylation staining (2x10 6 PBMCs) was performed using the Phosflow protocol (BD Biosciences). Cell sorting was performed on a FACSAria III instrument. Representative gating strategy is depicted in Supplementary Figure S3.
The number and purity of NK cells (median purity 99%) used in the RNA-seq is enlisted in Supplementary Table S4. RNA was extracted from purified NK cells (Qiagen RNeasy Micro Kit) and RNA integrity and quantity were checked with the Bioanalyzer 6000 pico chip (Agilent). Whole-transcriptome analysis with next-generation sequencing (RNA-seq), preparative techniques and statistical comparison (21) were performed by the VIB Nucleomics Core (KU Leuven, www.nucleomics.be). Detailed methods can be found in Supplementary File S5.
The number and purity of NK cells (median purity 99%) used in the RNA-seq is enlisted in Supplementary Table S4. RNA was extracted from purified NK cells (Qiagen RNeasy Micro Kit) and RNA integrity and quantity were checked with the Bioanalyzer 6000 pico chip (Agilent). Whole-transcriptome analysis with next-generation sequencing (RNA-seq), preparative techniques and statistical comparison (21) were performed by the VIB Nucleomics Core (KU Leuven, www.nucleomics.be). Detailed methods can be found in Supplementary File S5.
4
STAT1 Expression in T Cell Subsets
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Whole venous blood was collected into vacutainer tubes containing EDTA. STAT1 expression was studied following the BD Phosflow protocol as described above. STAT1 expression in FoxP3+ and FoxP3− Th lymphocytes was analysed using a panel of anti-CD45RA−FITC/anti-FoxP3−PE or just anti FoxP3−Alexa Flour 488 antibodies together with anti-CD45 PerCP/anti-STAT1−Alexa Flour 647/anti-CD4−Pe-Cy 7 antibodies in both cases. All antibodies were obtained from BD Biosciences. The samples were analysed on the LSR II (BD Biosciences) flow cytometer, equipped with a 488 nm solid-state laser and a 633 nm helium-neon laser. The analysis of data was performed using the FACSDiva software (BDBiosciences) and FlowJo (Tree Star).
5
PBMC Activation and Cytokine Signaling
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Peripheral blood mononuclear cells (PBMCs) were maintained for 24 hours in RPMI 1640 medium containing 0.1% serum. To activate cytokine-induced signalling, PBMCs were treated in RPMI 1640 containing TNF-α (100 ng/mL) for 5–30 min at 37°C. Then the cells were fixed and permeabilised according to the instructions of BD Phosflow Protocol. The treated and untreated cells were stained with antibody for 1 hour at room temperature. Stained cells were acquired by flow cytometry on a BD Accuri C6 flow cytometer (Becton Dickinson) and analysed using the FlowJo software (Tree Star).
6
Comprehensive Murine Immune Cell Analysis
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Composition of murine immune cells was analyzed using the following antibodies: CD3 (145-2C11; BioLegend), CD19 (6D5; BioLegend), CD20 (SA275A11; BioLegend), CD11b (M1/70; BioLegend), CD11c (N418; BioLegend), CD45 (30-F11; BioLegend), Ly6C (HK1.4; BioLegend) and Ly6G (1A8: BioLegend). B cell maturation was analyzed using the following antibodies: CD19 (6D5; BioLegend), CD21 (7G6; BD Bioscience), CD23 (B3B4; BD Bioscience), CD93 (AA4.1; BioLegend), CD45R/B220 (RA3-6B2; BioLegend), IgD (11-26c.2a; BioLegend) and IgM (AF6-78; BD Bioscience). Monocyte, macrophage and microglia activation, differentiation and molecules involved in antigen presentation were determined using: CD40 (3/23; BD Bioscience), CD68 (FA-11; BioLegend), CD69 (H1.2F3; BioLegend), CD80 (16-10A1; BioLegend), CD86 (GL-1; BioLegend), MHCII (AF6-120.1; BioLegend) and PD-L1 (MIH5; eBioscience). Fc receptors were blocked using monoclonal antibody specific for CD16/ CD32 (93; BioLegend). Dead cells were stained with a fixable viability kit (BioLegend). Samples were acquired on a BD LSR Fortessa (BD Bioscience). All data evaluation was performed using FlowJo software (FlowJo LLC, Ashland, USA).
Intracellular proteins were analyzed using the BD PhosFlow protocol and analyzed using the following antibodies: BTK (53/BTK; BD Bioscience), pBTK (N35-86, BD Bioscience), iNOS (W16030C, Biolegend) Arg1 (A1exF5, eBioscience).
Intracellular proteins were analyzed using the BD PhosFlow protocol and analyzed using the following antibodies: BTK (53/BTK; BD Bioscience), pBTK (N35-86, BD Bioscience), iNOS (W16030C, Biolegend) Arg1 (A1exF5, eBioscience).
7
Phosphorylation profiling of immune cells
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Phosphorylation of STAT5 was measured using the protocol developed by Goldeck et al. (22 (link)). Phosphorylation of the other proteins was analyzed using the BD Phosflow Protocol. PBMCs were incubated in TexMACS at 37°C/5% CO2 for 2 h, washed once at RT, and the final concentration was adjusted to 1 × 107 cells/mL. 106 PBMCs were incubated in a final volume of 100 µL in a 96-U bottom plate for 30′ at 37°C/5% CO2. Pre-warmed IL-15 was added at a final concentration of 10 ng/mL for 15′ at 37°C/5% CO2. Reaction was rapidly stopped by transferring the plate on ice and the addition of 100 µL of cold FACS buffer (pSTAT5) followed by the permeabilization and fixation with Cytofix/Cytoperm buffer (BD Biosciences) or by adding an equal volume of pre-warmed Cytofix Buffer for 10′ at 37°C/5% CO2 (other Phosflow Antibody). PBMCs were stained for cell-surface markers in Perm/Wash buffer for 30′ at 4°C before (pSTAT5) or after (other Phosflow Antibody) being further permeabilized by adding cold BD Perm Buffer III. Stability of the staining of cell-surface markers upon the use of BD Perm Buffer III was ensured in preliminary experiments.
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