cell trace violet (ctv) proliferation dye  (Thermo Fisher)

Journal: European Journal of Immunology

Article Title: Remote Force Modulation of the T‐Cell Receptor Reveals an NFAT‐Threshold for CD4 + T‐Cell Activation

doi: 10.1002/eji.202451716

Figure Lengend Snippet: Force application to TCR‐CD3, and not CD28, positively impacts T‐cell activation . Purified CD4 + T cells from Nr4a3 Tocky reporter mice were treated with 250 nm MNPs functionalised as indicated, subjected to 1 h of magnetic force, before assessment of Nr4a3 (A), CD69 (B) and CD25 (C) expression 4 h later. Force application driven through the TCR‐CD3 complex, but not CD28, positively impacts T‐cell signalling and proliferation (D, E) as evidenced through the comparison between MNPs coated with both anti‐CD3 and anti‐CD28 antibodies (CD3:CD28) and MNPs treated with anti‐CD3 alone together with soluble CD28 antibody treatment. All data are shown from a minimum of three independent experiments. (E) Representative histogram traces showing cell trace violet (CTV) dilution at 3 days post‐force application. Statistical significance was assessed via two‐way ANOVA with Sidak's post‐tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Purified naïve CD4+ T cells were loaded with 5 μM of Cell Trace Violet (CTV) proliferation dye as per manufacturer guidelines (Thermo Fischer, C34557 ).

Techniques: Activation Assay, Purification, Expressing, Comparison

Journal: Cell reports

Article Title: Salt Sensing by Serum/Glucocorticoid-Regulated Kinase 1 Promotes Th17-like Inflammatory Adaptation of Foxp3 + Regulatory T Cells

doi: 10.1016/j.celrep.2020.01.002

Figure Lengend Snippet: (A) Schematic diagram showing the in vitro co-culture system that enables the tracking of cell ontogeny (top panel). FACS-purified CD4 + GFP − Teff cells from B6.Foxp3 GFP mice were labeled with CellTrace Violet (CTV) proliferation dye and then combined with GFP + thymic Treg (tTreg) cells at a ratio of 6:1. After co-culture, cells of tTreg cell origin were identified as CTV − . Similarly, cells of original Teff cell origin were identified as CTV + . Representative flow cytometry plots showing the gating strategy to differentiate the cells of either tTreg or Teff cell origins (bottom panel). (B) tTreg and Teff cells were co-cultured (A) under increasing NaCl concentrations in the presence of Th17-polarizing cytokines for 72 h and in the presence or absence of a SGK1 inhibitor. Representative flow cytometry plots showing the expression of transcription factors RORγt and Helios in CTV − tTreg cells at 72 h. (C) Quantitation of the frequency of RORγt + cells in Foxp3 + CTV − tTreg cells. (D and E) Quantitative analyses of RORγt (D) and Foxp3 (E) protein expressions in tTreg cells by the assessment of median fluorescent intensity (MFI). (F) Representative flow cytometry plots showing the intracellular staining of IL-17A and RORγt in Teff cells or tTreg cells. (G) Assessment of the suppressive function of tTreg cells cultured in normal salt (NS) or high salt (HS) medium in a Th17-polarizing condition, as described in (A), in an in vitro suppression assay using CD4 + GFP − Teff cells as responders. Representative flow cytometry histograms of the proliferation of Teff by the dilution of the CTV proliferation dye. (H) Quantification of percent suppression at different Treg/Teff cell ratios. (I) Quantitative analysis of the frequency of RORγt + cells in Helios + and Helios − subsets of tTreg cells. Data are representative of three independent experiments with triplicates of each condition. Error bars represent mean ± standard deviation.

Article Snippet: FACS-sorted CD4 + CD45.1 + Foxp3 − (GFP − ) T EFF cells were labeled with Cell Trace Violet (CTV) Proliferation Dye (Invitrogen) and used as responder cells for the suppression assay.

Techniques: In Vitro, Co-Culture Assay, Purification, Labeling, Flow Cytometry, Cell Culture, Expressing, Quantitation Assay, Staining, Suppression Assay, Standard Deviation