Cell-culture reagents were purchased from Gibco Laboratories (Grand Island, NY, USA). Chemokines were purchased from PeproTech. Anti-CXCR7 antibody was purchased from Abcam; VCAM-1, GAPDH, and pERK/ERK, from Santa Cruz; pSTAT3, from BD Biosciences; F4/80, CD11b, CD206, cyclin D1 and Ki67 from NeoMarkers. All other reagents were of standard grade. The small-molecule CXCR7 antagonists were obtained from ChemoCentryx, Inc.; and STAT3 inhibitor (S31-201) was purchased from Calbiochem, Billerica, MA.
Pstat3
Manufactured by BD
Sourced in United States
The PSTAT3 is a laboratory equipment product manufactured by BD. It is a potentiostat, a device used for electrochemical measurements and analysis. The PSTAT3 provides a controlled potential and measures the resulting current in electrochemical experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pstat1, by BD (4 mentions)
Vcam 1, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Arginase 1, by BD (2 mentions)
Pstat5, by BD (2 mentions)
Chemokine, by Thermo Fisher Scientific (1 mentions)
P erk erk, by Santa Cruz Biotechnology (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Pstat5, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
Fix perm medium, by Thermo Fisher Scientific (1 mentions)
Icam 1, by BD (1 mentions)
Vla 4, by BD (1 mentions)
Cd11b, by BD (1 mentions)
Cd11c, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Cd62l, by BD (1 mentions)
Jwh 015, by Bio-Techne (1 mentions)
P egfr, by Santa Cruz Biotechnology (1 mentions)
P fak, by Abcam (1 mentions)
15 protocols using pstat3
1
CXCR7 Antagonists in Inflammation
2
Phospho-STAT Activation in MOG-Induced Cells
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Cells from the spleen and lymph nodes of MOG-immunized mice were cultured as described above. After 0, 15, 30 and 60 min, cells were fixed with 4% paraformaldehyde for 10 min, centrifuged at 1500 rpm for 5 min and permeabilized on ice with 100% cold methanol for another 10 min. Cells were then washed with PBS and stained with the following antibodies: anti-CD4 (eBioscience), pSTAT1 (BD Biosciences), pSTAT3 (BD Biosciences), pSTAT4 (eBioscience), pSTAT5 (eBioscience) and pSTAT6 (eBioscience) for flow cytometric analysis.
3
Multiparametric Flow Cytometry Immunophenotyping
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For surface-marker staining, cells were incubated with fluorochrome-conjugated Abs to CD4, CD8, CD11b, CD11c, CD80 CD86, VLA-4, ICAM-1, CD62L and CD44 (BD Biosciences, San Jose, CA) at the recommended dilution or isotype control Abs for 30 min on ice. To analyze MOG-specific Th1, Th2 and Th17 cells, splenocytes or CNS-infiltrating MNCs were stimulated with 25 μg/ml MOG peptide for 72 h or overnight, followed by stimulation with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of GolgiPlug for 5 h. Cells were surface-stained with mAbs against CD4 and CD8. Cells were then washed, fixed, and permeabilized with Fix & Perm Medium (Invitrogen), and intracellular cytokines were stained with Abs against IL17, IFN-γ, or IL4 (BD Biosciences). For phosphorylated STAT staining, cells were fixed with 4% paraformaldehyde for 10 min at 37 °C, permeabilized with 90% methanol for 30 min on ice, and stained with p-STAT1, p-STAT3, p-STAT4 and CD4 mAbs (BD Biosciences, San Jose, CA). Foxp3 staining was carried out using a commercial kit, according to the manufacturer’s instructions (eBioscience, San Diego, CA). Flow cytometric analysis was performed on FACSAria (BD Biosciences, San Jose, CA) and data were analyzed with FlowJo software (Treestar, Ashland, OR).
4
Signaling Pathways Analysis in Cell Lines
5
Protein Isolation and Quantification
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The total protein was isolated from myeloid cells by a protein lysis buffer (50 mM Tris–HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, and 0.5% Nonidet P-40; containing a protease inhibitor cocktail; Sigma, St Louis, MO), as described before [48 (link)] and quantified using BCA (Bicinchononic Acid assay kit; Thermo Fisher, Rockford, IL). An equal amount of protein was separated by 10–12% SDS polyacrylamide gel electrophoresis and transferred onto the polyvinylidene difluoride membrane and blocked with 5% skimmed milk. iNOS, Arginase 1, STAT-3, pSTAT3 were purchased from BD Biosciences (San Jose, CA). IL-1β and DLST were purchased from Cell Signaling Technology (Danvers, MA). S100A9 and CSF-R were purchased from Proteintech (Rosemont, IL, USA). The dilutions used for the antibodies are summarized in Table S3 .
6
Immunoblotting and Luminex Assays of Stat3
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For immunoblotting, total protein concentration was measured within M-PER lysates using the Coomassie assay kit (Bio-Rad, Hercules, CA) with BSA as a standard. Total cell lysates containing 20 µg of total protein were separated by 7.5% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA in 0.1% TBS-T (0.1% Tween-20 in TBS) for 1 h and incubated overnight at 4°C in blocking buffer with one of the primary antibodies against total Stat3, pStat3 (BD-Biosciences), β-actin, GAPDH, CCT1, CCT2, and CCT5 (Abcam); washed; incubated with appropriate secondary antibodies conjugated to horseradish peroxidase; washed; and developed with ECL substrate (Thermo Scientific). For Luminex bead assays, cells were lysed and assayed for pStat3, total Stat3, and GAPDH using Millipore (Milliplex) kits as described by the manufacturer. Levels of each protein analyte were determined and analyzed using the Bio-Plex suspension array system (Bio-Rad).
7
Profiling Immune Cell Subsets with Phospho-STAT Signaling
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PBMCs were stained with fixable viability dye Zombie Aqua at room temperature for 10 min, washed twice in FACS buffer (3% FBS in PBS1 ×) followed by staining for the following surface markers before fixation and permeabilization: CD3 (11–0036-42, Invitrogen, FITC), CD4 (25–0047-42 or 47–0049-42, Invitrogen, PE-Cy7 or APC-Cy7), CD8 (17–0086-42 or 25–0088-42, Invitrogen, APC or PE-Cy7). Cells were fixed with BD cytofix for 10 min at 37 °C. Afterwards, permeabilization was performed using BD phosflow Perm III (558,050, BD Biosciences) on ice for 30 min followed by intracellular staining with p-STAT3 (560,312, BD Biosciences, Pacific Blue, pY705) or p-STAT4 (17,186,668, ThermoScientific, APC, pY693) for 60 min at 4 °C.
8
Establishing Humanized Murine Models for HNSCC
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Antibodies against anti-human CD3, CD4, CD8, CD11c, CD11b, CD14, CD15, CD16, CD19, CD33, CD34, CD45, CD56, NKP46, CD123, arginase-1, neutrophil lipocalin, IFNγ, HLA-DR, and pStat3 were purchased from BD Biosciences, Biolegend, or ebiosciences. Synthetic 2’-5’ linked [R,R] dithio cyclic di-AMP (ML-RR-CDA) was a gift from Aduro Biotech. NOD.scid.IL2Rγcnull (NOG) mice were crossed to NOD.HLA-A2*0201 traNSGenic mice (Jackson) to generate NOD/SCID/γcnull HLA-A2+ (NOG-A2) mice in our lab and housed according to JHH Animal Care. In some cases, HSCFTL mice [NOD-Cg-PrKdcscidIL2γgtm1sug/JicTac mice engrafted with human CD34+ hematopoietic stem cells (HSCs) (Taconic), NSG, or NSG-HLA-A2.1 mice (Jackson) were used. Human CD34 microbead kit and pan T-cell isolation kit were purchased from Miltenyi Biotec. Human HNSCC cell lines used consisted of Cal27 and SCC25 (ATCC). The cell lines were authenticated by a short tandem repeat profiling analysis using the AmpFISTR Identifier PCR Amplification kit (Applied Biosystems) at the Genetic Resources Core Facility, JHH [43 (link)]. Cells were maintained in DMEM medium typically supplemented with 10% FBS and penicillin (100 U/mL) and streptomycin (100 mg/mL) and maintained in a humidified incubator at 37°C in a 5% CO2 atmosphere.
9
Flow Cytometric Analysis of Signaling Pathways
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As previously described by our team25 (link), cells were washed then fixed in 1.5% paraformaldehyde for 20 min at room temperature. Cells were re-rinsed and permeabilized using a 90% methanol/PBS solution. This step was realized on ice for a total period of 20 min. An additional wash cycle was performed, and cells were labeled for 30 min using the following fluorescence conjugated primary antibodies: pSTAT1 (BD Biosciences, AB_1645373), pSTAT3 (BD Biosciences, AB_647232), pSTAT5 (BD Biosciences, AB_399858), pERK1/2 (BD Biosciences, AB_399857), phosphorylated p38 (BD Biosciences, AB_399856) and Phospho NF-κB p65 (Thermofisher, AB_2572751). Cells were washed for the last time and flow cytometry analysis using the BD Accuri C6 Plus flow cytometer allowed estimation of ERK1/2, NF-κB, STAT1, STAT3, STAT5 and P38 phosphorylation levels. The histogram subtraction technique was applied based on Overton subtraction and thus the overlay of the histograms of interest. The analysis was then realized by the FCS express De Novo software42 (link).Three biological replicates were carried out.
10
Isolation and Activation of CD4+ T Cells
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PBMCs were isolated as described before and erythrocytes were depleted. Next, CD4+ T cells were isolated with magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec). CD4+ T cells were incubated in RPMI 1640 Glutamax (Gibco) supplemented with 100 µM β-mercaptoethanol (Life Technologies) for 60 min 37°C and then stained extracellularly for CD4. Afterwards, 1×106 cells were taken up in 500 µL prewarmed RPMI 1640 Glutamax (Gibco), supplemented with 100 U/mL penicillin/streptavidin (Gibco) and 10% FCS (Sigma) and incubated either with or without 20 ng/mL IL-23, 20 µg/mL anti-IL6 (BD Biosciences) and 2 µg/mL anti-IL22 (R&D) for 5 min at 37°C. Stimulation was stopped and cells were fixed by addition of cold 4% paraformaldehyde in phosphate buffered saline (PBS) and incubated for 10 min at room temperature. After a single wash with PBS, cells were permeabilised in 70% ice-cold methanol in PBS for 30 min on ice. The cells were then stained intracellularly for 30 min at room temperature with an antibody specific for phosphorylated STAT3 (pSTAT3) (BD Biosciences no. 557815) and analysed by flow cytometry.
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