CD14+ monocytes were isolated from HLA-matched donor peripheral blood by flow sorting using CD14-FITC (Becton Dickinson) and Moflo Astrios EQ flow cytometry sorter. Post-sorted cells were incubated in various conditions for 96 hours on ultra-low attachment plates (Corning). 811-conditioned media (811 CM) was collected off 90% confluent 811 cell line and immediately frozen at −80°C for later use. IL6 neutralizing antibody (R& D Systems) was used at concentration of 50µg/ml and tocilizumab (Genentech) was used at concentration of 1mg/ml. Media were harvested from cells after 96 hours and cytokine concentrations were measured by multiplex bead cytokine analysis as described above. Cells were stained with pSTAT3-PE (BD) according to manufacturer’s instructions and run on Beckman Coulter Gallios 561; analysis was done using FlowJo v10.0.7 (FlowJo).
Pstat3 pe
Manufactured by BD
Sourced in United States
The PSTAT3-PE is a laboratory equipment product. It is designed to measure and analyze various physical and chemical properties of samples. The core function of the PSTAT3-PE is to perform potentiostatic measurements, which are used to study electrochemical processes and properties.
Automatically generated - may contain errors
Lab products found in correlation
Pstat5 pe, by BD (3 mentions)
Perm buffer 3, by BD (2 mentions)
Cd14 fitc, by BD (1 mentions)
Tocilizumab, by Genentech (1 mentions)
Il 6 neutralizing antibody, by R&D Systems (1 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Facscalibur, by BD (1 mentions)
Cd3 bv510, by BioLegend (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Cd4 bv421, by BioLegend (1 mentions)
Cd19 pe cy7, by BioLegend (1 mentions)
Igg1 fc, by R&D Systems (1 mentions)
Atr 107, by Pfizer (1 mentions)
Recombinant human il 21, by Thermo Fisher Scientific (1 mentions)
Cytofix buffer, by BD (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Lyse fix, by BD (1 mentions)
Interleukin 7 (il 7), by R&D Systems (1 mentions)
Cd3 percp, by BD (1 mentions)
Pd 1 pe, by Thermo Fisher Scientific (1 mentions)
6 protocols using pstat3 pe
1
CD14+ Monocyte Profiling of IL-6 Signaling
2
IL-6 and IL-7 Signaling Pathway Assay
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For each subject, blood samples were collected by venipuncture into 2‐mL sodium heparin tubes before and at 1, 6, and 12 hours after administration of upadacitinib (day 1 in Study 1; days 1 and 14 in Study 2, Part 1; days 3 and 28 of Study 2, Part 2), tofacitinib (days 1 and 14 in Study 2, Part 3), or placebo (day 1 in Study 1; days 1 and 14 in Study 2, Part 1; days 3 and 28 of Study 2, Part 2). The blood samples were frozen and shipped to the AbbVie Bioresearch Center (Worcester, Massachusetts) for processing and analysis. IL‐6–induced pSTAT3 and IL‐7–induced pSTAT5 were assayed as previously described.19 Briefly, recombinant human IL‐6 or IL‐7 (R&D Systems, Minneapolis, Minnesota) was added to blood followed by addition of surface antibodies (CD14‐APC, CD3‐fluorescein isothiocyanate; BD Biosciences, San Jose, California), sample lysis, wash, and resuspension in BD Perm buffer III (BD Biosciences). Samples were then washed and stained with pSTAT5‐PE or pSTAT3‐PE (BD Biosciences) and analyzed on a FACSCalibur (BD Biosciences). The percent change was calculated as a change in pSTAT3 and pSTAT5 at 1, 6, and 12 hours following drug administration divided by the baseline measurement at t = 0 for each subject multiplied by 100.
3
STAT3 Phosphorylation in T and B Cells
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Phosphorylation of STAT3 by CD4+ T cells and CD19+ B cells was determined by phospho-specific flow cytometry. In brief, PBMCs were stained with CD3 BV510 (Biolegend) and CD19 Pe-Cy7 (Biolegend) for 30 min at RT in the dark. Next, the cells were incubated for 30 min at 37°C with various concentrations of the humanized anti-IL-21R antibody ATR-107 (Pfizer) or isotype-matched control (IgG1-Fc, R&D systems) followed by stimulation with recombinant human IL-21 (100 ng/ml, eBioscience) or recombinant human IL-6 (100 ng/ml, PeproTech, Rocky Hill, NJ, USA) for 15 min at 37°C. Cells were fixed for 10 min with Cytofix buffer (BD Biosciences) at 37°C and permeabilized 30 min in 1 ml methanol 90% at −20°C. Next, samples were stained with CD4 BV421 (Biolegend) and pSTAT3 PE (BD Biosciences). STAT3 phosphorylation was calculated as the median fluorescence intensity.
4
IFN-α Induced STAT Activation Assay
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PBMCs were surface stained with CD19, CD24, and CD38 antibodies in ice-cold PBS for 30 min, then rested for 1 hr in RPMI medium. Cells were stimulated by incubating with 10,000 U/mL exogenous IFN-α (PBL) for 1 or 5 min and then fixed with formaldehyde for 10 min at 37°C, washed with ice-cold PBS, and permeabilized with 90% ice-cold methanol for 20 min on ice. tSTAT1-PE, tSTAT3-AF647, pSTAT1-AF647, and pSTAT3-PE (BD Phosflow) were added to cells, for 1 hr at 37°C.
5
Measurement of STAT Phosphorylation in Blood
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For each subject, blood was collected by venipuncture into 2 mL sodium heparin tubes at 0, 1, 6, and 12 h post upadacitinib or tofacitinib dose. Recombinant human IL-6 (400 ng/ml), or IL-7 (400 ng/ml), (R&D Systems, Minneapolis, MN) was added to blood and incubated for 10 min at 37°C. Surface antibodies were added (CD14-APC, CD3-fluorescein isothiocyanate [FITC]; BD Biosciences, San Jose, CA) and incubated on ice for an additional 20 min. Samples were lysed (Lyse/Fix, BD Biosciences, San Jose, CA) and incubated for 10 min at 37°C. Samples were washed and stored at − 70°C. For intracellular staining, samples were thawed, washed, and resuspended in BD Perm buffer III (BD Biosciences, San Jose, CA) on ice for 30 min. Samples were washed and stained with pSTAT5-PE or pSTAT3-PE (BD Biosciences, San Jose, CA) for 60 min at room temperature and then analyzed immediately on a FACSCalibur. Geometric means were determined using FlowJo analysis software. Percent inhibition of relevant STAT phosphorylation was calculated as follows: (1-(Induction of pSTAT at 1 h – baseline pSTAT at 0 h) / (Induction of pSTAT at 0 h – baseline pSTAT at 0 h)*100.
6
Immunophenotyping of T Cell Subsets
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To determine the percentage of T cell subsets, heparin-anticoagulated whole blood were collected and stained with CD3-PerCP (BD Bioscience, New Jersey, US), CD4-FITC (BD Bioscience, New Jersey, US), CXCR5-APC (Biolegand, California, US), PD-1-PE (eBioscience, California, US), ICOS-PE (eBioscience, California, US) and CD25-APC (BD Bioscience, New Jersey, US). After fixed and permeabilized, samples were stained with FoxP3-PE (BD Bioscience, New Jersey, US), p-STAT3-PE (BD Bioscience, New Jersey, US), p-STAT5-PE (BD Bioscience, New Jersey, US) and p-STAT4-PE (BD Bioscience, New Jersey, US). After stimulation with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) (Sigma-Aldrich, US), ionomycin (1 μg/ml) (Sigma-Aldrich, US), and Golgi stop (BD Bioscience, New Jersey, US) for 5 h, the fixation and permeablication were performed. Then samples were stained with IL-21-PE (BD Bioscience, New Jersey, US). Samples were measured with FACS Canto II (BD Biosciences, New Jersey, US). Gating strategy used for the analysis of all immune parameters was shown in Additional file 1 .
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