I am stimulating with IL-27, IL-6, or IL-21 and trying to see pSTAT3 in naive CD4+ T cells. I have tried BD's protocol using the Perm buffer III, I have used Stumhofer et al., 2007, AOH Hall et al., 2012 and a combination of the 3 of them, I have also tried the Ab in different colors. I am incubating with complete IMDM media (done it in plates or eppendorf) for short periods of time (5, 15, 20, 30 min), with or without TCR stimulation, I have fixed at 37°C and -20°C, permeabilized with 90% methanol 30min on ice, or -20°C overnight, or using the Perm III buffer. I appreciate any tips you can give me, thank you.
Hello!
For exaple, Fixed cells were washed in Stain Buffer (BD Biosciences), vortexed and permeabilized by adding 1 ml of chilled Phosflow™ Perm Buffer III (and incubating for 30 min on ice. Samples were then washed and incubated with the mouse anti-human antibodies (all from BD Biosciences) anti-CD3-PE-Cy5 (clone UCHT1;), -CD4-FITC (clone RPA-T4), -CD8-FITC (clone RPA-T8), and -phosphorylated (p)-STAT3-PE (Phosflow™) at room temperature for 60 min. Then, the cells were washed and resuspended in Stain Buffer (BD Biosciences), and were subjected to flow cytometry analysis on a FACSCalibur instrument (BD Biosciences). To analyze the mean fluorescence intensity (MFI) of p-STAT3 in peripheral CD4+ and CD8+ T cells, cells were gated in CD3+ T/CD4+ T and CD3+ T/CD8+ T regions.
Article Aberrant expression of p-STAT3 in peripheral blood CD4(+) an...
Flow Cytometry
Permeabilized cells were thawed and washed three times with staining buffer (PBS/2%FBS/0.5%BSA) to remove methanol. Cells were resuspended in the same volume of staining buffer and staining antibodies were added. For assessment of pSTAT1 and pSTAT3, the following staining panel was utilized: CD3-V450 (UCHT1), CD4-PerCP-Cy5.5 (SK.3), CD45RA-PE-Cy7 (L48), CD8-V500 (RPA-T8), CD16-PE (3G8), CD20-PerCP-Cy5.5 (H1), CD33-PE-Cy7 (P67.6), pSTAT1 (pY701)-AF647 (4a), and pSTAT3 (pY705)-AF488 (4/P-STAT3) antibodies (BD Biosciences, San Jose, CA, USA). Naïve CD4+ T cell population was determined by the following markers: CD3+CD4+CD45RA+. The magnitude of each individual's pSTAT1 and pSTAT3 response to IL-6 was expressed as the IL-6 induced median fluorescence intensity (MFI) minus the unstimulated MFI for pSTAT1 and pSTAT3.
For assessment of intracellular total STAT1 and STAT3 levels, LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies, Carlsbad, CA, USA) was added to unstimulated cells for 30 minutes and washed away prior to PFA fixation and permeabilization. After washing the cells to remove methanol, the following staining panel was utilized: CD3-V500 (UCHT1), CD4-PerCP-Cy5.5 (SK.3), CD8-PacBlue (RPA-T8), CD45RA-PE-Cy7 (L48), CD62L-FITC (DREG-56), STAT1-AF647 (1/Stat1), and STAT3-PE (M59-50) antibodies (BD Biosciences). For assessment of gp130 and IL-6Rα expression levels on live PBMCs, the following staining panel was utilized: CD3-V500 (UCHT1), CD4-PerCP-Cy5.5 (SK.3), CD8-PacBlue (RPA-T8), CD45RA-PE-Cy7 (L48), CD62L-e605NC (DREG-56), gp130-PE (AM64) (BD Biosciences), and IL-6Rα-AF647 (BL-126) (Biolegend, San Diego, CA, USA) antibodies, and LIVE/DEAD Fixable Blue Dead Cell Stain (Life Technologies). Expression of total STAT1, STAT3, gp130 and IL-6Rα was expressed by subtracting the MFI of isotype stains.
Flow cytometry was performed using FACS Canto, LSR II, or Fortessa Flow Cytometers (BD Biosciences). Flow cytometry data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). t tests were used to determine the statistical significance of BC patient with healthy donors (Graphpad Prism, GraphPad Software, LaJolla, CA, USA).
Article IL-6 Signaling in Peripheral Blood T Cells Predicts Clinical...
Alexandr Chernov pretty much those are the protocols I have tried, thank you
A stupid idea... maybe the difficulty is to find a good positive control with high enough Stat3 signaling to set up the best staining condition. In naive T cells, the amount of receptors and signaling might be low. What about activating the cells for a few days in a Th17-like differentiation protocol and then wash, and measure STAT3 signaling with or without cytokine stimulation? Might be too much work.
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