ionomycin  (Valiant)

Journal: Scientific Reports

Article Title: T cell signaling and Treg dysfunction correlate to disease kinetics in IL-2Rα-KO autoimmune mice

doi: 10.1038/s41598-020-78975-y

Figure Lengend Snippet: IL-2Rα-KO Tregs acquire effector functionality. ( A ) Representative flow plots, and frequency of CD4 and CD8 Tregs in the spleen in 19-day-old WT and IL-2Rα-KO mice. n = 4–11 mice per experimental group. Representative flow plots and frequency of T cells producing IL-2 and IFNγ in 19-day-old WT and IL-2Rα-KO LN T cells ( B1 , B2 ) or Tregs ( C ) post 5-h PMA and ionomycin stimulation. n = 4–6 mice per experimental group. ( D ) CTLA-4 expression on CD4 Treg cells at day 19 by mean fluorescent intensity (MFI). ( E ) Frequency of IL-10+ 19-day-old WT and IL-2Rα-KO CD4 Treg cells post 5-h PMA and ionomycin stimulation. ( F ) Frequency of CTLA-4+FoxP3+CD4 T cells or CTLA-4+FoxP3− CD4 T cells are shown for 19-day-old WT and IL-2Rα-KO mice. ( G ) Frequency of IL-10+ 19-day-old WT and IL-2Rα-KO CD8 T and CD8 Treg cells post 5-h PMA and ionomycin stimulation. n = 4–6 mice per experimental group over 4 independent experiments. Statistics: unpaired Student’s t test; *p

Article Snippet: Lymphocytes and splenocytes were stimulated for 5 h at 37 °C with 70 ng/mL phorbol 12-myristate 13-acetate (PMA; Fisher Scientific) and 700 ng/mL ionomycin (MP-Biomedicals) and treated with 5 μg/mL brefeldin A (MP-Biomedicals) .

Techniques: Mouse Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells

doi: 10.1084/jem.20032177

Figure Lengend Snippet: CCR8 + T cells predominate in normal human skin. Analyses were performed with freshly isolated cells. (a) Flow cytometric detection of CCR8 (gray histograms) and CCR9 (bold line) in T cells isolated from human skin, lamina propria of small intestine, and peripheral blood. Peptide-blocked control for CCR8 staining is shown as a thin line. CD4, CD8, and CD3 denote the respective populations analyzed within the αβTCR + cell gate, and the numbers indicate percentages of CCR8- and CCR9-positive cells, respectively. Data for skin and intestinal cells are representative for nine and three donors, respectively. (b) Chemotactic migration of skin T cells, gated on CD4 + cells (black bars) and CD8 + (white bars), in response to chemokines as indicated. Values represent the percentage of migrated cells as a proportion of input cells, and control denotes the level of migration in the absence of chemokines. (c) Expression of CLA and CD45RA on CCR8 + skin T cells. Gates were set for CD4 + αβTCR + cells (CD4) and CD8 + αβTCR + cells (CD8), and the numbers refer to the percentage of cells within each quadrant. (d) Production of various cytokines by CD4 + (black bars) or CD8 + (white bars) CCR8 + αβTCR + skin T cells. Intracellular accumulation of cytokines in response to PMA/ionomycin was measured by flow cytometry and is expressed as percent cytokine-positive cells. Chemotaxis and cytokine production data are representative of four independent experiments.

Article Snippet: Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry.

Techniques: Isolation, Flow Cytometry, Staining, Migration, Expressing, Cytometry, Chemotaxis Assay

Journal: The Journal of Experimental Medicine

Article Title: A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells

doi: 10.1084/jem.20032177

Figure Lengend Snippet: Analysis of T cell clones derived from skin CCR8 + T cells. Sorted CCR8 + T cells isolated from normal skin were cloned under nonpolarizing conditions. (a) For cytokine and (b) CCL1 secretion analysis, 6 CD4 + and 26 CD8 + T cell clones were stimulated with PMA/ionomycin for 24 h, and cell-free supernatants were tested in ELISA. (c) Expression of CCR8 by T cell clones, as determined by flow cytometry. Percent (%) CCR8 + refers to the fraction of CCR8-positive cells present within individual clones. (d) Detailed analysis of three clones representing high, intermediate, and low level expression of CCR8. Cells stained with anti-CCR8 antibodies (left, shaded histogram) or peptide-blocked anti-CCR8 (unshaded histogram). Numbers in parentheses indicate the clone number, whereas numbers above the gate lines refer to the percentage of cells positive for CCR8. The corresponding center panels show chemotactic responses of the same clones toward CCL1, expressed as the number of migrated cells counted (mean of triplicate wells ± SEM) in five high-power fields. Background migration in the absence of chemokine is indicated by open circles. The expression of CLA versus CD45RA, with the percentage of cells positive within each quadrant indicated (right).

Article Snippet: Sorted αβTCR+ CCR8+ CD8+ or αβTCR+ CCR8+ CD8− skin T cells, or skin T cell clones, were stimulated with 10 ng/ml PMA, 1 μg/ml ionomycin, and 10 μg/ml brefeldin A (Qbiogene) for 4.5–10 h. After fixing with 2% paraformaldehyde for 20 min at 4°C and permeabilization with 0.2% saponin/2% FCS in PBS− , cells were stained with antibodies and analyzed by flow cytometry.

Techniques: Clone Assay, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Staining, Migration

Journal: PLoS ONE

Article Title: Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

doi: 10.1371/journal.pone.0147742

Figure Lengend Snippet: BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with PMA/Ionomycin and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.

Article Snippet: NK cell treatment NK cells were either analyzed directly after the enrichment or were stimulated for 4hrs with 50ng/ml phorbol 12-myristate 13-acetate (PMA; Acros Organics, Fisher Scientific) and 1μg/ml ionomycin (MP Biomedicals).

Techniques:

Journal: PLoS ONE

Article Title: Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

doi: 10.1371/journal.pone.0147742

Figure Lengend Snippet: Overview of sample collection and processing. (A) Study design and sample collection. Details of the complete study have been published previously [ 1 ]. (B) Blood samples were stained for total leukocyte populations or used for NK cell enrichment. NK cells were analyzed for surface marker expression or cytokine production either naive or stimulated with PMA and ionomycin (Iono). Half of the peripheral blood mononuclear cells (PBMCs) were frozen and used later for the cytotoxicity assay.

Article Snippet: NK cell treatment NK cells were either analyzed directly after the enrichment or were stimulated for 4hrs with 50ng/ml phorbol 12-myristate 13-acetate (PMA; Acros Organics, Fisher Scientific) and 1μg/ml ionomycin (MP Biomedicals).

Techniques: Staining, Marker, Expressing, Cytotoxicity Assay

Journal: The Journal of Cell Biology

Article Title: Nuclear calcium is required for human T cell activation

doi: 10.1083/jcb.201602001

Figure Lengend Snippet: Nuclear calcium controls the expression of activation markers and cytokines in primary human T cells. T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry either were left unstimulated (ctr) or were stimulated with a combination of antibodies to CD3 and CD28 or with a combination of PMA and ionomycin. Expression of CD25 (A), CD69 (B), IL-2 (C), and IFNγ (D) in T cells was analyzed by flow cytometry with FITC-conjugated antibodies. Dot plots (left) are representative examples of three independent experiments; histograms (right) show the mean percentage of FITC-positive cells (blue dots) from three independent experiments. Error bars represent SEM. Statistically significant differences are indicated with asterisks (*, P

Article Snippet: Samples were run for an additional 9 min. At the end of the analysis, 1 µM ionomycin (MP Biomedicals) was added to obtain the maximum signal.

Techniques: Expressing, Activation Assay, Transfection, Flow Cytometry, Cytometry

Journal: The Journal of Cell Biology

Article Title: Nuclear calcium is required for human T cell activation

doi: 10.1083/jcb.201602001

Figure Lengend Snippet: Analysis of nuclear calcium–regulated human T cell adaptogenomics. Representation of gene profiling data of human T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 2, 6, and 16 h. (A) Heatmap with cluster dendrogram of 939 genes differentially expressed (log2 fold change) in at least one condition. Maximum value is set to 2.0. Fold ratio values greater than or equal to 2.0 are depicted with saturated red; values less than or equal to −2.0 are depicted with saturated green. (B and C) Number of genes induced by nuclear calcium signaling (i.e., down-regulated by CaMBP4.GFP; B) and repressed by nuclear calcium signaling (up-regulated by CaMBP4; C) at different time points on the basis of Illumina Human Sentrix-12 BeadChip arrays. (D–G) Validation of gene regulatory events by QRT-PCR. Gene expression was analyzed in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 2 h ( Egr2 and Egr3 ) and 6 h ( DUSP2 and Ikzf5 ) or were left unstimulated (ctr). mRNA levels are expressed relative to those in GFP-expressing T cells stimulated with PMA and ionomycin, which was set to 100% ( n = 3). Statistically significant differences are indicated with an asterisk (*, P

Article Snippet: Samples were run for an additional 9 min. At the end of the analysis, 1 µM ionomycin (MP Biomedicals) was added to obtain the maximum signal.

Techniques: Transfection, Expressing, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Nuclear calcium is required for human T cell activation

doi: 10.1083/jcb.201602001

Figure Lengend Snippet: Nuclear calcium signaling does not regulate nuclear translocation of NFATc1 in primary human T cells. (A) T cells transfected with expression vectors for CaMBP4.mCherry (CaMBP4) or mCherry were left unstimulated (ctr), were stimulated with a combination of antibodies to CD3 and CD28 in the presence or absence of cyclosporine A (CsA) and FK506, or were stimulated with ionomycin (iono) for 30 min. Cells were stained with antibodies against NFATc1. Nuclei were stained with Hoechst. Bar, 5 µm. (B) Quantitative analysis of NFATc1 nuclear localization ( n = 3). Error bars represent SEM.

Article Snippet: Samples were run for an additional 9 min. At the end of the analysis, 1 µM ionomycin (MP Biomedicals) was added to obtain the maximum signal.

Techniques: Translocation Assay, Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Nuclear calcium is required for human T cell activation

doi: 10.1083/jcb.201602001

Figure Lengend Snippet: Nuclear calcium signaling differentially regulates CREB activation and CREM expression in primary human T cells. (A, left) Immunoblot analysis of CREB and the serine 133–phosphorylated form of CREB (pCREB) in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP and stimulated with a combination of PMA and ionomycin for 30 or 120 min. A representative example is shown. (right) Quantitative analysis of three experiments; fold induction is relative to the pCREB/CREB ratio in unstimulated GFP-expressing T cells. (B) QRT-PCR analysis of Crem mRNA in T cells transfected with expression vectors for CaMBP4.GFP (CaMBP4) or GFP. Cells were stimulated with a combination of PMA and ionomycin for 6 h or were left unstimulated (ctr). mRNA levels are expressed relative to the mRNA level in GFP-expressing T cells stimulated with a combination of PMA and ionomycin, which was set to 100%. n = 3. Statistically significant differences are indicated with an asterisk (*, P

Article Snippet: Samples were run for an additional 9 min. At the end of the analysis, 1 µM ionomycin (MP Biomedicals) was added to obtain the maximum signal.

Techniques: Activation Assay, Expressing, Transfection, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Nuclear calcium is required for human T cell activation

doi: 10.1083/jcb.201602001

Figure Lengend Snippet: TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum ( F max ) and minimum ( F min ) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (Δ F / F 0 ) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.

Article Snippet: Samples were run for an additional 9 min. At the end of the analysis, 1 µM ionomycin (MP Biomedicals) was added to obtain the maximum signal.

Techniques: Activation Assay, Transfection, Fluorescence