For immunofluorescence staining of whole blood and PBMC, antibodies included CD45-ECD (Beckman Coulter, Indianapolis IN), CD3-e780, CD4-e450, CD8-AF700, CD15-FITC (eBioscience, San Diego, CA), CD45-FITC, CD4-PerCP-Cy5-5, CD14-PECy7, HLA-DR-APC, HLA-DR-BV421, CD8-APC-H7, CCR7-BV605, CD25-BV605, CD45RA-PECy7, CD45RO-APC-H7, CCR4-PECy7, CD38-APC, CD127-BV650 (BD Bioscience, San Jose, CA), CD14-AF700, and CD3-AF700 (Biolegend, San Diego, CA). For phosphoSTAT staining, antibodies included CD3-BV785 (Biolegend), CD4-BV605, CD8-BV510, CD14-PECy7, CD19-BV421, CD16-BV650, pSTAT3-647, pSTAT5-PE (BD Biosciences), and pSTAT1-488 (Cell Signaling Technologies, Danvers, MA). Cell types were identified according to the criteria of the Human Immunology Project [19 (link)], and a full list of antibodies used to identify individual cell types including those not reported here can be found in Additional file 2 : Table S1. Hematological toxicity was graded according to NCI CTCAE v4.0 guidelines and is reported in Additional files 3 and 4 : Tables S2 and S3.
Pstat5 pe
Manufactured by BD
Sourced in United States
PSTAT5-PE is a fluorescent-labeled antibody for the detection and analysis of phosphorylated STAT5 protein, a key signaling molecule involved in cellular processes, using flow cytometry. The product provides a tool for researchers to monitor STAT5 phosphorylation status in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Pstat3 pe, by BD (3 mentions)
Lsrfortessa, by BD (2 mentions)
Perm buffer 3, by BD (2 mentions)
Cd8 apc h7, by BD (1 mentions)
Cd14 pe cy7, by BD (1 mentions)
Cd45 fitc, by Thermo Fisher Scientific (1 mentions)
Cd25 bv605, by BD (1 mentions)
Cd8 bv510, by BD (1 mentions)
Cd38 apc, by BD (1 mentions)
Hla dr apc, by BD (1 mentions)
Cd15 fitc, by Thermo Fisher Scientific (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Cd45ra pe cy7, by BD (1 mentions)
Cd3 e780, by Thermo Fisher Scientific (1 mentions)
Cd8 af700, by Thermo Fisher Scientific (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd4 e450, by Thermo Fisher Scientific (1 mentions)
Cd45ro apc h7, by BD (1 mentions)
Cd3 af700, by BioLegend (1 mentions)
Cd19 bv421, by BD (1 mentions)
8 protocols using pstat5 pe
1
Immunophenotyping of Whole Blood and PBMCs
2
Monocyte STAT5 Phosphorylation Assay
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Monocytes were enriched from the bone marrow of WT and DKI mice as described above. Monocytes were seeded at a density of 1 x 105 cells/well in a 96-well plate in complete medium and were rested at 37°C for 30 min. The monocytes were stained with CD11b-FITC (N418), Ly6C-PE (HK1.4), and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher L34976) for 20 min at 37°C. Following surface staining, the monocytes were stimulated with 100 ng/mL GM-CSF (Peprotech 315-03) for 15 min at 37°C. The stimulated monocytes were then fixed in 2% paraformaldehyde in PBS at 37°C for 10 min. Following fixation, the monocytes were permeabilized with 90% ice cold methanol in PBS and were incubated at -20°C for 30 min. Monocytes were washed with staining buffer, then stained with pSTAT5-PE (BD Biosciences 47/Stat5 (pY694)) at room temperature for 30 min. The monocytes were washed and resuspended in staining buffer. Cells were analyzed on a BD LSRFortessa.
3
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.
4
Phosphorylation of STAT5 in BMMNCs
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BM mononuclear cells (BMMNCs) were isolated by Ficoll-Hypaque density gradient centrifugation (750 g × for 5 min at 20°C; density, 1.077; Tianjin Hao Yang Biological Products Technology Co., Ltd., Tianjin, China). The samples were stimulated with BMMNCs (2–3×106cells) G-CSF (100 ng/ml) or SCF (100 ng/ml) to induce the phosphorylation of STAT5 and fixed with crosslinking reagent (2% paraformaldehyde; Sigma-Aldrich, St. Louis, MO, USA). To stabilize the phosphorylation, the cells were fixed using 2% paraformaldehyde in PBS for 10 min at 4°C. The monoclonal antibodies CD34-PerCP and CD59-FITC were then added to the samples. After incubating at 4°C for 30 min, RBCs in the samples were lysed with 2 ml RBC lysing solution. The cells were mixed with 1.0 ml of FACS™ permeabilizing solution (BD Biosciences) for 10 min in the dark, and then incubated with STAT5-PE (cat no., 562077; 1:100 dilution) and p-STAT5-PE (cat no., 129010; 1:100 dilution) mouse monoclonal antibodies (BD Biosciences) at 4°C for 30 min. The cells were then washed twice with PBS. Information about ≥100,000 cells was acquired for each sample by FCM.
5
Treg-NK Cell Interaction Dynamics
6
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.
7
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 .
8
Flow Cytometric Analysis of pSTAT5 Expression
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Ficoll-enriched BM cells were stained with LIVE/DEAD Blue (ThermoFisher), blocked with FC block (BD Biosciences) and stained with CD45-Amycan (clone: 2D1, BD Biosciences), CD33-BUV395 (clone: P67.6, BD Biosciences), and FLT3 (CD135)-BV421 (clone: 4G8, BD Biosciences). Cells were then fixed with BD Cytofix buffer (BS Biosciences) and permeabilized with BD phosflow perm buffer III (BD Biosciences). Cells were then stained with pSTAT5-PE (clone: 47/Stat5, BD Biosciences). Sample analysis was performed in the OSUCCC Flow Cytometry Shared Resource and Immune Monitoring & Discovery Platform using the Cytek Aurora. Data were analyzed using FCS Express v.7 (De Novo Software).
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