Purified M-MDSCs and G-MDSCs from the spleen of CT-26 tumor bearing mice were stained for intracellular IFN-γ according to manufacturer’s instructions (BD Pharmingen). For STAT1 and 3 phosphorylation, MDSCs were treated with IFN-γ for 15 minutes and fixed with 2% paraformaldehyde for 10 minutes at room temperature and permeabilized with 100% ice cold methanol for 30 minutes at 4°C. The cells were washed and stained with anti-phospho STAT1 (pY701) or anti-phospho STAT3 (pY705) antibodies (BD Pharmingen) for 30 minutes.
Anti phospho stat1 py701
Manufactured by BD
Anti-phospho STAT1 (pY701) is a primary antibody that specifically recognizes the tyrosine 701 phosphorylated form of the STAT1 protein. STAT1 is a key transcription factor involved in cellular signaling pathways.
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2 protocols using anti phospho stat1 py701
1
Quantifying MDSC Cytokine Signaling
2
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.
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