The following antibodies were used for Phosflow and IL-6R phenotype analysis in samples processed at Newcastle: anti-CD3-Pacific blue (UCHT1), anti-CD19-FITC (4G7), anti-CD19-APC (HIB19), anti-Stat3 (pY705)-Alexa Fluor 647 (4/P-STAT3) and anti-Stat1 (pY701)-Alexa Fluor 647 (4a; all BD Biosciences, Oxford, UK); anti-CD4-APC-eFluor 780 (SK3; eBioscience, Hatfield, UK); IL-6R-Fluorescein (17506; R&D Systems Europe, Abingdon, UK). Phosflow was performed on whole blood, which was either left unstimulated or stimulated with 100 ng/mL IL-6 (PeproTech EC, London, UK) for 15 min at 37°C. BD Phosflow Lyse/Fix and BD Phosflow Perm Buffer III (both BD Biosciences) were used as per the manufacturers’ instructions. IL-6R expression was assessed in whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturers’ instructions. Data were collected on a BD FACSCanto II (BD Biosciences) and analysed using FlowJo (Treestar, Ashland, Oregon, USA). The protocol followed for samples processed in Brisbane was similar, using anti-CD3-Pacific blue (UCHT1), and anti-Stat3 (pY705)-PE (4/P-STAT3; BD Biosciences), with a Gallios flow cytometer and Kaluza software for data acquisition/analysis (both Beckman Coulter). Flow-Set Pro Fluorospheres (Beckman Coulter) were used to normalise median fluorescence intensities (MFI) for pSTAT3 measurements between data sets.
Phosflow lyse fix
Manufactured by BD
Phosflow Lyse/Fix is a laboratory product designed for the preparation of cells for flow cytometry analysis. It is used to lyse and fix cells, preserving cellular structure and phosphorylated proteins for subsequent immunostaining and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phosflow perm buffer 3, by BD (3 mentions)
Human trustain fcx fc blocking solution, by BioLegend (2 mentions)
Facs symphony, by BD (2 mentions)
Zombie near ir reagent, by BioLegend (2 mentions)
Anti cd3 pacific blue, by BD (1 mentions)
Anti cd4 apc efluor 780 sk3, by Thermo Fisher Scientific (1 mentions)
Flow set pro fluorospheres, by Beckman Coulter (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Facs lysing solution, by BD (1 mentions)
Kaluza software, by Beckman Coulter (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facscanto 2, by BD (1 mentions)
Pstat5, by BD (1 mentions)
Perm buffer 3, by BD (1 mentions)
Stain buffer, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
X vivo 15, by Lonza (1 mentions)
Live dead fixable dead cell stain, by Thermo Fisher Scientific (1 mentions)
Biotinylated anti cd3 clone 145 2c11, by BD (1 mentions)
9 protocols using phosflow lyse fix
1
Phosflow and IL-6R Phenotype Analysis
2
Dissecting T Cell Activation in Tumor Microenvironment
3
Immunophenotyping and Signaling Analysis
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BM and splenic cells were prepared and analyzed as described previously (Guitart et al., 2017 (link); Guitart et al., 2013 (link); Kranc et al., 2009 (link); Mortensen et al., 2011 (link); Paris et al., 2019 (link); Vukovic et al., 2016 (link)) followed by c-Kit enrichment (described below). Cells were incubated with Fc block and stained with biotin-conjugated anti-Lineage marker antibodies (anti-CD4, anti-CD5, anti-CD8a, anti-CD11b, anti-B220, anti-Gr-1, and anti-Ter119), APC-Cy7–conjugated anti-c-Kit, and Pacific Blue–conjugated anti-Sca-1. Biotin-conjugated Lineage markers were then stained with PerCP-conjugated Streptavidin. Cells were then fixed and permeabilized using Phosflow Lyse/Fix, Phosflow Perm Buffer III, and stain buffer (all from BD Biosciences) according to the manufacturer’s instructions. After processing, cells were stained with AF647-conjugated anti-pStat1 or anti-pStat3.
4
Cytokine-Stimulated HSC Analysis
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Bone marrow cells from different animals were harvested and pre-cultured in medium (X-Vivo15, Lonza) without cytokines for 10 minutes and stimulated with cytokines (stem cell factor, SCF at 1 ng/ml, R&D Systems; and thrombopoietin, TPO, at 4 ng/ml, R&D Systems) for the time indicated. Subsequently, the cells were fixed and permeabilized using Phosflow Lyse/Fix and Perm/Wash buffers (BD Biosciences) according to manufacturer's instructions. Cells were then stained for surface markers to define the HSC-containing population as described above and with antibodies against pERK1/2-FITC (1∶20; Cell Signaling).
5
T Cell Activation and Signaling Analysis
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Cells from spleen and LNs of CalDAG GEFI–/–, CalDAG GEFI+/– and CalDAG GEFI+/+ mice were stained for dead cells using the LIVE/DEAD Fixable Dead Cell Stain (Invitrogen) and subsequently for surface markers CD4 and CD25. Next, cells were coated with 10 µg/ml biotinylated anti-CD3 (clone 145-2C11, BD Biosciences) and 5 µg/ml biotinylated anti-CD28 (clone 37.51, BD Biosciences) for 15 min on ice. After removal of excess antibody, cells were pre-warmed to 37 °C and stimulation was initiated by addition of 10 µg/ml streptavidin in cRPMI. After indicated time points, cells were fixed (BD Biosciences Phosflow Lyse/Fix) and permeabilized (BD Biosciences Phosflow Perm Buffer III) according to the manufacturer’s protocol. Cells were stained intracellularly for ERK1/2 pT202/pY204 or isotype control (both BD Biosciences Phosflow) and Foxp3 at 4 °C overnight.
6
PBMC Stimulation and Intracellular Staining
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Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-blood by Ficoll centrifugation. Intracellular monocyte staining: 1 × 106 PBMC were stimulated for 15 min at 37 °C with IFN-α (500 U/ml; Miltenyi Biotec) or IFN-γ (100 ng/ml; Miltenyi Biotec). Intracellular CD4 staining: PBMC were first stained with anti-CD4 (Ancell; clone QS4120) and then stimulated at 37 °C with either IL-27 (200 ng/ml) or IFN-α (10,000 U/ml) for 7.5 min, 15 min and 30 min. Stimulation was stopped by adding lysis and fixation buffer (Phosflow lyse/fix; BD) followed by incubation for 10 min at 37 °C. Cells were permeabilized for 30 min on ice with pre-cooled Phosflow PermIII buffer (BD) prior to staining with anti-phospho-STAT1 (pY701) (BD; clone 4a) and anti-CD14, when monocytes were studied (Beckman Coulter; clone RMO52). Data acquisition was done using either Navios flow cytometer (Beckman Coulter) or BD LSR II (BD Biosciences). FlowJo v7.2.5/v10.0.7 (Treestar) was used for data analysis.
7
Multiparametric Flow Cytometry of Whole Blood
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Multiparametric flow cytometry analysis of whole blood and airway cells was standardized across study visits using the acquisition setting automatic calibration built into the BD software on the BD FACS Symphony instrument which provides constant and robust output from the flow cytometer over time. Samples were pre-stained for 10 minutes on ice in the dark with the Human TruStain FcX Fc blocking solution and the Zombie near IR reagent (Biolegend), then stained for surface markers (see Table S4 ). Cells were washed, fixed in Lyse/Fix Phosflow (BD Biosciences) and acquired on a BD FACS Symphony (BD Biosciences). Analysis and compensation were performed in FlowJo V10.6.2 (BD Biosciences).
8
Multiparametric Flow Cytometry Analysis
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Multiparametric flow cytometry analysis of whole blood and airway cells was standardized across study visits using the acquisition setting automatic calibration built into the BD software on the BD FACSymphony instrument (BD Biosciences), which provides constant and robust output from the flow cytometer over time. Samples were prestained for 10 minutes on ice in the dark with the Human TruStain FcX Fc blocking solution and the Zombie near-IR reagent (BioLegend), and then stained for surface markers (see Supplemental Table 4 for antibodies). Cells were washed, fixed in Lyse/Fix Phosflow (BD Biosciences), and acquired on a FACSymphony. Analysis and compensation were performed in FlowJo v10.6.2 (BD Biosciences). Image cytometry was performed as previously described (45 (link)) (see Supplemental Methods ).
9
Comprehensive Immune Cell Profiling
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Airway cells obtained from the BAL were resuspended in cold PBS-EDTA (2.5 mM) and incubated for 10 minutes on ice in the dark with a mouse Fc block (BioLegend) and the Zombie Near-IR Live/Dead stain (BioLegend). After the preincubation, cells were stained for 20 minutes on ice in the dark for surface expression of CD45 (clone 30-F11), CD11c (clone N418), Ly6G (clone 1A8), Ly6C (clone HK1.4), CD11b (clone M1/70), CD4 (clone GK1.5), CD8 (clone 53-6.7), NK1.1 (clone S17016D) and CD3 (clone 145-2C11) (all antibodies were purchased from BioLegend). Cells were then washed 3 times with cold PBS-EDTA and then fixed overnight at 4°C with the Lyse/Fix Phosflow (BD Biosciences). The next day, the fixative was removed following a 500g, 4°C centrifugation for 10 minutes, and cells were resuspended in cold PBS-EDTA. Cell acquisition was performed on a BD FACS Symphony (BD Biosciences), and data were analyzed using FlowJo v10.7 (BD Biosciences).
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