Computational protocol: Altered regulatory T-cell fractions and Helios expression in clinically isolated syndrome: clues to the development of multiple sclerosis

Similar protocols

Protocol publication

[…] T cells were analysed using combinations of Fixable Viability Stain 780, anti-human CD3 BV510 (HIT3a), CD4 BB515, APC-H7 or Alexa Fluor 700 (all RPA-T4), CD8 PerCP-Cy5.5 (SK1), CD14 BV650 (M5E2), CD25 PE-Cy7 (M-A251), CD127 BV421 (HIL-7R-M21), CD45RA Alexa Fluor 700 (HI100), CD146 BV421 (P1H12), CD161 PE (DX12), C-C chemokine motif receptor 6 (CCR6; BV650 (11A9), CCR7 BV786 (3D12), CXCR3 Pe-Cy7 (1C6/CXCR3), CXCR5 BB515 or Alexa Fluor 647 (both RF8B2), HLA-DR PE-Cy7 (G46-6), FoxP3 PE-CF594 (259D/C7), Helios Alexa Fluor 647 (22F6), IL-6 PE-CF594 (MQ2-13A5), IL-10 BV786 (JES3-9D7), IL-17A PE (N49-653) (henceforth referred to as IL-17) and IFN-γ Alexa Fluor 647 (4S.B3) (all from BD Biosciences, San Jose, CA, USA). Analysis of B cells was performed using CD45 FITC (H130), CD19 PE (HIB19), CD24 PE-CF594 (ML5), IgD Pe-Cy7 (IA6-2), CD20 APC-H7 (2H7), CD38 BV421 (HIT2) and CD27 BV510 (L128). For cytokine detection, PBMCs were activated with Leukocyte Activation Cocktail with GolgiPlug (BD Biosciences), containing phorbol 12-myristate 13-acetate, Ionomycin and Brefeldin A, for 4 h at 37 °C in a 5% CO2 atmosphere. Surface staining was conducted using Brilliant Stain Buffer (BD Biosciences) (T cells) or phosphate-buffered saline containing 4% foetal bovine serum (B cells), and the eBioscience FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA) was used for intracellular staining. The fluorescence minus one method was used to confirm gating in populations with high background fluorescence, and PBMCs from a single independent control subject were analysed on six independent occasions to monitor the repeatability of geometric MFI. Data were acquired using a BD LSRFortessa (BD Biosciences), and postacquisition analysis was conducted using the FlowJo V10.1 software (Tree Star, Ashland, OR, USA). [...] For parametric data, differences between groups were determined by Student’s t-tests, and mean and s.d. are reported. For non-parametric data, Mann–Whitney U-tests were used with median and interquartile range reported. Correlations of continuous parametric and non-parametric variables were measured by the Pearson (r) and Spearman (rho) correlation coefficients, respectively. Coefficient of variation is reported for purposes of quantifying test–retest reliability. To determine the level of agreement between T-cell panels, intraclass correlation estimates were calculated based on a mean-rating, absolute-agreement, two-way mixed-effects model. For all tests, the alpha level was set at <0.05. Statistical analyses were performed using IBM SPSS Statistics for Windows, version 23 (IBM Corp., Armonk, NY, USA) and figures were generated in GraphPad Prism, version 6 (GraphPad, La Jolla, CA, USA). […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry