multiplex cytokine detection kit  (Bio-Tech Pharmacal Inc)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Identification of Distinct Human TH17 Cell Subsets and Generation of Stable TH17 Clones from PBMC for Functional Characterization. A Schematic representation of the workflow to generate T H 17-IL22 + /IFNg + and T H 17-IL-10 + clones used to perform bulk ATAC-seq and RNA-seq data sets. In brief, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using density gradient centrifugation. The samples were enriched for CD4 + CCR6 + CXCR3- TH17 cells, referred to as “bulk TH17 cells.” Viable IL-17-producing cells were isolated by flow cytometry following a 3-hour stimulation with PMA and ionomycin using a IL-17 capture assay. The single TH17 cell clones were sorted into 384-well plates and expanded with allogeneic γ-irradiated feeder cells and phytohemagglutinin in complete medium containing IL-2. After approximately ten days, clones were transferred to 96-well plates for expansion, and following 2–3 weeks, their cytokine profiles were analyzed. T cell clones were then evaluated at two stages: day 0 (resting state) and day 5 (activated state). On day 5, they were stimulated for 48 hours with anti-CD3 and CD28, followed by an additional 3 days in uncoated plates. On both evaluation days, cells underwent further stimulation — 5 hours for protein analysis and 2 hours for RNA and chromatin-accessibility (ATAC-seq) analysis. Only TH17 clones exhibiting a stable cytokine profile after two rounds of resting and reactivation were selected for RNA-seq and ATAC-seq analysis. B Intracellular staining of IL-17 and IFNγ (top) and IL-22 and IL-10 (bottom) in a T H 17-IL10 + clone (right) and a T H 17-IL22 + /IFNg + clone (left) in the resting state (Day 0) and 5 days post-activation (Day 5). Numbers in quadrants indicate percent cells. C Frequency of IL-17+, IL-10+, IFNγ+, and IL-22+ cells among 6 independent TH17-IL-22 + /IFNγ + (left) and TH17-IL-10 + (right) clones at Day 0 and Day 5. Each symbol represents an individual T cell clone ( n = 6); data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01 (one-way ANOVA). TH17 clones were selected for RNA and ATAC-seq analysis based on the following criteria: ≥50% IL-17A+ cells at Day 0, ≥15% IL-22+ cells at Day 0 and Day 5, ≥15% IFNγ+ cells at Day 0 and Day 5 for TH17-IL-22 + /IFNγ + clones; ≥50% IL-17A+ cells at Day 0, ≥15% IL-10+ cells at Day 5 for TH17-IL-10 + clones

Article Snippet: Viable IL-17-producing bulk TH17 cells were sorted by flow cytometry using the IL-17 cytokine secretion assay (Miltenyi Biotec#130–094–536) following 3 h of stimulation with phorbol 12-myristate 13-acetate (PMA) (0.2 μM) and ionomycin (1 μg/ml) (both from Sigma-Aldrich), according to the manufacturer’s instructions.

Techniques: Clone Assay, Functional Assay, RNA Sequencing, Isolation, Gradient Centrifugation, Flow Cytometry, Irradiation, Staining, Activation Assay