Immature MDDCs (1 × 106 cells per ml) were incubated at 37 °C, 5% CO2 for 2 h, and then treated with either recombinant IL-6 (R&D Systems) at 20 ng ml−1 or a 1 : 2 dilution of TCM for 15 min. Cells were fixed with Cytofix buffer (BD Biosciences), permeabilized with Phosflow Perm Buffer III (BD Biosciences) and pSTAT3 expression was measured using phospho-STAT3 (BD Biosciences) following the recommended protocol. Samples were acquired on a FACScalibur flow cytometer.
Phospho stat3
Manufactured by BD
Sourced in United States
Phospho-STAT3 is a laboratory equipment product that is used to detect and quantify the phosphorylated form of the STAT3 protein. STAT3 is a transcription factor that plays a key role in cellular signaling pathways. The Phospho-STAT3 product allows for the measurement of the activated, phosphorylated state of STAT3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosflow perm buffer 3, by BD (1 mentions)
Recombinant il 6, by R&D Systems (1 mentions)
Cytofix buffer, by BD (1 mentions)
Phosflow fix buffer 1, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Recombinant human bdnf, by R&D Systems (1 mentions)
Phospho stat1, by BD (1 mentions)
Stat1, by BD (1 mentions)
3 protocols using phospho stat3
1
Analyzing pSTAT3 in Dendritic Cells
2
Phospho-STAT activation in MPN mice
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Protocol adapted from (Kalaitzidis and Neel, 2008 (link)). In brief, lineage-depleted bone marrow cells from 10–14-mo-old wild-type and Nol3−/− MPN mice were incubated in IMDM 2% FBS for 30 min before cytokine stimulation. Cells were then stimulated with 50 ng/ml TPO, 10 ng/ml G-CSF, or PBS (unstimulated) for 15 min. After stimulation, cells were fixed using equal volume of Phosflow Fix Buffer I (BD) for 10 min at 37°C. Cells were pelleted by centrifugation for 5 min at 300 g, and after removal of supernatant, permeabilized using ice-cold acetone dropwise and incubated for 10 min on ice. Cells were then washed twice with 2% FBS in PBS, and stored in 2% FBS in PBS at 4°C until flow cytometry analysis. Samples were analyzed at the same time using a BD LSR II flow cytometer and antibodies against phospho-STAT5 (BD) and phospho-STAT3 (BD).
3
Investigating Schwann Cell Signaling Pathways
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Four cell lines were all purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), including human neuroblastoma BE(2 (link))-C and SH-SY5Y, rat Schwann cell (RSC) RT4-D6P2T, and human Schwann cell (HSC) from ScienCell Research Lab (San Diego, CA, USA). Following the manual, the cell culture medium and related reagents were used, and all were purchased from the cell culture facility at University of California, San Francisco. Recombinant human BDNF was purchased from R&D systems Inc. (Minneapolis, MN, USA) and used for the treatment. S100, p75 antibodies (Chemicon, Inc., Temecula, CA, USA) and secondary antibody FITC-conjugated goat anti-mouse IgG (Chemicon, Inc., Temecula, CA, USA) were used for immunofluorescence staining. Beta-actin, JAK2, phospho-JAK2 (Chemicon, Inc., Temecula, CA, USA), STAT1, phospho-STAT1, STAT3, and phospho-STAT3 (BD Biosciences, CA, USA) antibodies were used for immunoblot. The reagents in ECL kit (Amersham Life Sciences Inc., Arlington Heights, IL, USA) were used as substrate for all the immunoblotting. OSM-M and IL-6 enzyme linked immunosorbent assay (ELISA) kit (R&D systems Inc., Minneapolis, MN, USA) were applied to assay the secretion of cytokine from Schwann cells.
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