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  • 99
    Thermo Fisher ionomycin
    Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca 2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS 473 -Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca 2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca 2+ stores, uninfected cells were treated with 1 μM <t>ionomycin.</t> The mean intracellular Ca 2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 10 4 cells; the asterisk indicates a significant increase in Ca 2+ concentration relative to the mock-infected control ( P
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam ionomycin
    Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in <t>ionomycin</t> permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P
    Ionomycin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson ionomycin
    Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and <t>ionomycin,</t> showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P
    Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 8343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
    Ionomycin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Calbiotech ionomycin
    IFN-γ-mediated protection prevents IL-17-mediated mortality. (A) Number of CD4 + T cells in the infiltrating population and distribution of Thy1.1 positive cells measured by flow cytometry at day eight p.i. Data represent means (±SD) of twelve mice per group combined from three separate experiments. (B) IL-17 mRNA expression determined by quantitative real-time PCR in infected SCID recipients of WT, WT/GKO or GKO CD4 + T cells. Data represent the mean of two experiments with n = 4 in each group per experiment. (C) Splenocytes of immunized GKO donors cultured in the presence of JHMV with or without recombinant IFN-γ (10 ng/ml) for six days and restimulated four hours with <t>PMA/Ionomycin.</t> Intracellular cytokine expression on CD4 + T cells analyzed by flow cytometry using FITC-IFN-γ, PE-IL-17 and the corresponding isotype controls. Dot plots are representative of duplicates from two separate experiments. (D) Survival of infected SCID recipients of WT/GKO (n = 6), WT/GKO + anti-IFN-γ mAb (n = 8) and GKO (n = 4) CD4 + T cells assessed daily. Data are representative of two separate experiments.
    Ionomycin, supplied by Calbiotech, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical ionomycin
    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or <t>Ionomycin</t> ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).
    Ionomycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem ionomycin
    Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and <t>ionomycin</t> (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.
    Ionomycin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM ionomycin
    Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + <t>Ionomycin</t> for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P
    Ionomycin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories ionomycin
    LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM <t>ionomycin.</t> D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P
    Ionomycin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA ionomycin
    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore <t>ionomycin</t> was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.
    Ionomycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology ionomycin
    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM <t>ionomycin</t> (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
    Ionomycin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Selleck Chemicals ionomycin
    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM <t>ionomycin</t> (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
    Ionomycin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris ionomycin
    AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + <t>Ionomycin</t> and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P
    Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    InvivoGen ionomycin
    T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, <t>ionomycin</t> (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate
    Ionomycin, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend ionomycin
    Siglec-9 blockade restores cytokine secretion and degranulation of NK cells in chronic hepatitis B (CHB) patients. PBMCs from CHB patients pretreated with Siglec-9 blocking antibody or isotype control IgG were stimulated with PMA plus <t>ionomycin.</t> Then, the expression of IFN-γ (A) , TNF-α (B) , and CD107a (C) were analyzed by flow cytometry. The right panels showed the representative flow cytometry data from one subject.
    Ionomycin, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LKT Laboratories ionomycin
    TAT-CBD3 inhibits the forward mode of NCX. Neurons were loaded with Fura-2FF. A , 5 μ m <t>ionomycin</t> in combination with 20 μ m AP-5 was applied to neurons. Where indicated, neurons were subjected to Na + /NMDG replacement ( B ) or treated with
    Ionomycin, supplied by LKT Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore ionomycin
    ATRA increases CD8+ T cell activation. (A) Representative flow cytometric gating strategy showing contour plots analyzing intracellular cytokine staining in unstimulated cells or cells stimulated for 5 hours with PMA + <t>ionomycin.</t> CD8(+) T cells were identified by first gating on single, live, CD3(+)CD8(+) cells. (B) Comparison of the pre-treatment and post-treatment frequency of CD8(+)IFNγ (+)CD107a(+) T cells. (C) Correlation between the frequencies of MDSCs and CD107a(+)IFNγ(+)CD8(+) T cells. Error bars indicate standard error of the mean. * Denotes p
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 46801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Alomone Labs ionomycin
    Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with <t>ionomycin</t> (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).
    Ionomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Avantor ionomycin
    1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and <t>ionomycin</t> in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.
    Ionomycin, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Biogems International ionomycin
    1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and <t>ionomycin</t> in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.
    Ionomycin, supplied by Biogems International, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Mannheim ionomycin
    Quantification of amplitude of intercellular calcium signals and extent in cultured astrocytes. Analysis of intercellular calcium signaling generated ( A ) by mechanical stimulation ( n = 12) and ( B ) by focal application of <t>ionomycin</t> ( n = 21). Relative amplitude of [Ca 2+ ] i increases ( left scales , open columns ) and number of unresponsive cells ( right scales , dashed line ) were plotted by identifying the cellular row from which they were recorded. Cells were classified according to their location in reference to the stimulated cell, numbered 0 . Typically, stimulation was performed in the center of a microscopic field composed of ∼30 cells forming approximately six to seven cellular rows. As the distance from the stimulated cell increased, the amplitude of the response decreased, and more cells did not respond. For both modes of stimulation, the extent of intercellular calcium signaling generally exceeded the investigated cell population.
    Ionomycin, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific ionomycin
    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and <t>ionomycin</t> in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
    Ionomycin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co ionomycin
    TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L <t>ionomycin</t> or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P
    Ionomycin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant ionomycin
    BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with <t>PMA/Ionomycin</t> 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.
    Ionomycin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Adipogen ionomycin
    Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with <t>ionomycin</t> (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.
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    Image Search Results


    Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca 2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS 473 -Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca 2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca 2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca 2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 10 4 cells; the asterisk indicates a significant increase in Ca 2+ concentration relative to the mock-infected control ( P

    Journal: Journal of Virology

    Article Title: Herpes Simplex Virus Type 2 Glycoprotein H Interacts with Integrin αvβ3 To Facilitate Viral Entry and Calcium Signaling in Human Genital Tract Epithelial Cells

    doi: 10.1128/JVI.00725-14

    Figure Lengend Snippet: Integrin αvβ3 contributes to the virus-induced cytoplasmic Ca 2+ response post-Akt phosphorylation. (a) CaSki cells transfected with siIntegrinαvβ3 RNA were mock infected (serum free medium) or infected with purified HSV-2(G) (10 PFU/cell), and cell lysates were prepared for Western blot analysis at the indicated times p.i. Blots were incubated with anti pS 473 -Akt123 and then stripped and probed with anti-total Akt123. A representative blot from 3 independent experiments is shown. (b) CaSki cells were loaded with Calcium Green 72 h posttransfection with the indicated siRNA and synchronously mock infected or infected with purified HSV-2(G) (5 PFU/cell). Live images were acquired 3 min after a temperature shift to 37°C. Cellular membranes were stained with CellTrace (red), nuclei were stained with Hoechst (blue), and Ca 2+ is green. Representative XYZ images from 3 independent experiments are shown. Bars = 9.2 μm. (c) Transfected CaSki cells were loaded with fura-2, infected with purified HSV-2(G) (2 PFU/cell), or mock infected. To assess whether siRNA transfections impacted the intracellular Ca 2+ stores, uninfected cells were treated with 1 μM ionomycin. The mean intracellular Ca 2+ concentration (nM) over 1 h was calculated from 4 wells, each containing 5 × 10 4 cells; the asterisk indicates a significant increase in Ca 2+ concentration relative to the mock-infected control ( P

    Article Snippet: The cells were then transferred to a SpectraMaxMFe temperature-regulated chamber at 37°C (Molecular Devices) without washing; photometric data for [Ca2+ ] were generated by exciting cells at 340 and 380 nm and measuring emission at 510 nm every minute for 1 h. An intracellular calibration was performed with each experiment by determining the fluorescence ratio (340:380) in the presence of Ca2+ -free 10 mM K2 -EGTA buffer ( R min ) and 10 mM Ca-EGTA buffer containing 10 μM ionomycin ( R max ) (C-3008, calcium calibration buffer kit no. 1; Invitrogen Molecular Probes).

    Techniques: Transfection, Infection, Purification, Western Blot, Incubation, Staining, Concentration Assay

    Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in ionomycin permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P

    Journal: Nitric oxide : biology and chemistry

    Article Title: Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells

    doi: 10.1016/j.niox.2017.09.001

    Figure Lengend Snippet: Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in ionomycin permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P

    Article Snippet: Ionomycin was obtained from Abcam (Cambridge, UK).

    Techniques: Derivative Assay

    Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P

    Journal: Nature immunology

    Article Title: The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR

    doi: 10.1038/s41590-018-0137-8

    Figure Lengend Snippet: Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P

    Article Snippet: For intracellular cytokine staining, cells were stimulated with PMA and ionomycin in the presence of BD GoligiStop (BD Bioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, In Vitro, Enzyme-linked Immunosorbent Assay

    Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Transfection, Proliferation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Transfection, Activity Assay

    Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activity Assay, Transfection, FACS, Enzyme-linked Immunosorbent Assay

    Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    IFN-γ-mediated protection prevents IL-17-mediated mortality. (A) Number of CD4 + T cells in the infiltrating population and distribution of Thy1.1 positive cells measured by flow cytometry at day eight p.i. Data represent means (±SD) of twelve mice per group combined from three separate experiments. (B) IL-17 mRNA expression determined by quantitative real-time PCR in infected SCID recipients of WT, WT/GKO or GKO CD4 + T cells. Data represent the mean of two experiments with n = 4 in each group per experiment. (C) Splenocytes of immunized GKO donors cultured in the presence of JHMV with or without recombinant IFN-γ (10 ng/ml) for six days and restimulated four hours with PMA/Ionomycin. Intracellular cytokine expression on CD4 + T cells analyzed by flow cytometry using FITC-IFN-γ, PE-IL-17 and the corresponding isotype controls. Dot plots are representative of duplicates from two separate experiments. (D) Survival of infected SCID recipients of WT/GKO (n = 6), WT/GKO + anti-IFN-γ mAb (n = 8) and GKO (n = 4) CD4 + T cells assessed daily. Data are representative of two separate experiments.

    Journal: Journal of Neuroinflammation

    Article Title: IFN-? protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils

    doi: 10.1186/1742-2094-9-104

    Figure Lengend Snippet: IFN-γ-mediated protection prevents IL-17-mediated mortality. (A) Number of CD4 + T cells in the infiltrating population and distribution of Thy1.1 positive cells measured by flow cytometry at day eight p.i. Data represent means (±SD) of twelve mice per group combined from three separate experiments. (B) IL-17 mRNA expression determined by quantitative real-time PCR in infected SCID recipients of WT, WT/GKO or GKO CD4 + T cells. Data represent the mean of two experiments with n = 4 in each group per experiment. (C) Splenocytes of immunized GKO donors cultured in the presence of JHMV with or without recombinant IFN-γ (10 ng/ml) for six days and restimulated four hours with PMA/Ionomycin. Intracellular cytokine expression on CD4 + T cells analyzed by flow cytometry using FITC-IFN-γ, PE-IL-17 and the corresponding isotype controls. Dot plots are representative of duplicates from two separate experiments. (D) Survival of infected SCID recipients of WT/GKO (n = 6), WT/GKO + anti-IFN-γ mAb (n = 8) and GKO (n = 4) CD4 + T cells assessed daily. Data are representative of two separate experiments.

    Article Snippet: Cytokine production from both splenic cultures or ex vivo lymph node cells was measured following four hours stimulation with PMA (10 ng/ml) (Acros Organics, Geel, Belgium) and ionomycin (1 μM) (Calbiotech, Spring Valley, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Infection, Cell Culture, Recombinant

    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Journal: bioRxiv

    Article Title: A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis

    doi: 10.1101/669325

    Figure Lengend Snippet: TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Article Snippet: Drugs and reagents Lipopolysaccharides (LPS) from S. enterica serotype Typhimurium (Sigma-Aldrich, L6143-1MG, 10ug/ml): Peptidoglycan (PGN) from S. aureus (Invivogen, tlrl-pgns2): Monophosphoryl Lipid A (MPLA-SM) (Invivogen, tlrl-mpla): ML-SA1 (Sigma Aldrich, SML0627) Ionomycin (Cayman Chemical Company, 10004974): FK-506 (Cayman Chemical Company, 10007965): BAPTA AM (Cayman Chemical Company, 15551): Ned-19 (Cayman Chemical Company, 17527): Kuromanin (Cayman Chemical Company, 16406): Apigenin (Cayman Chemical Company, 10010275): CCCP (Cayman Chemical Company, 25458): N-acetyl-L-Cysteine (NAC) (Cayman Chemical Company, 20261): N-acetyl-L-Cysteine amide (NACA) (Cayman Chemical Company, 25866): Apocynin (Cayman Chemical Company, 11976): D609 (Cayman Chemical, 13307, 50 µM): kb NB 142-70 (Cayman Chemical Company, 18002).

    Techniques: Activation Assay, Infection

    Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.

    Journal: FEBS letters

    Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

    doi:

    Figure Lengend Snippet: Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.

    Article Snippet: Ionomycin was purchased from Alexis Biochemicals (San Diego, CA).

    Techniques: Activation Assay

    Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.

    Journal: FEBS letters

    Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

    doi:

    Figure Lengend Snippet: Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.

    Article Snippet: Ionomycin was purchased from Alexis Biochemicals (San Diego, CA).

    Techniques: Activation Assay, Incubation, Western Blot

    Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + Ionomycin for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P

    Journal: PLoS ONE

    Article Title: Conditional Deletion of TAK1 in T Cells Reveals a Pivotal Role of TCRαβ+ Intraepithelial Lymphocytes in Preventing Lymphopenia-Associated Colitis

    doi: 10.1371/journal.pone.0128761

    Figure Lengend Snippet: Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + Ionomycin for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P

    Article Snippet: For intracellular cytokine staining, cells were cultured with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Wako) plus 500 ng/ml ionomycin (Wako) for 5 hours in the presence of GolgiStop (BD Bioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Amplification

    LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM ionomycin. D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx

    doi: 10.1152/ajpcell.00298.2015

    Figure Lengend Snippet: LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM ionomycin. D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P

    Article Snippet: Key reagents and their sources were as follows: Escherichia coli LPS serotype O1101:B4 (List Biological Laboratories), nigericin (NG) (Sigma-Aldrich), PR-619 (Sigma-Aldrich), ionomycin (LC Laboratories), H-Leu-Leu-OMe·HBr (Bachem), CA-074-methyl ester (Bachem), Imject Alum (Pierce), monosodium urate (Invivogen), disuccinimidyl suberate (DSS; Sigma-Aldrich), anti-caspase-1 (p20) mouse mAb (Casper-1; Adipogen), anti-NLRP3 mouse mAb (Cryo-2; Adipogen), and anti-NLRP3 rat mAb (Clone 768319; R & D Systems).

    Techniques: Fluorescence, Incubation

    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Journal: PLoS ONE

    Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

    doi: 10.1371/journal.pone.0063521

    Figure Lengend Snippet: Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Article Snippet: Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany).

    Techniques: Chemotaxis Assay, Purification, Migration, Flow Cytometry, Cytometry, Negative Control, Positive Control

    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Journal: Scientific Reports

    Article Title: Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii

    doi: 10.1038/srep42746

    Figure Lengend Snippet: Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Article Snippet: Images were taken every 10 seconds for a total of 5 minutes and 2 μM ionomycin (Santa Cruz Biotechnology) was added after 2–4 time points were imaged.

    Techniques: Fluorescence, Standard Deviation, Microscopy

    AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Journal: PLoS ONE

    Article Title: mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

    doi: 10.1371/journal.pone.0154564

    Figure Lengend Snippet: AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Article Snippet: To determine the percentage of TH1 and TH17 cells, mononuclear cells were stimulated with PMA (50ng/mL, sigma) and Ionomycin (1μg/mL, Tocris) and BFA (1:1000, eBioscience) for 6 hours.

    Techniques: In Vitro, Isolation, Mouse Assay, Labeling, Staining, Expressing

    T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, ionomycin (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate

    Journal: Cell Death and Differentiation

    Article Title: Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

    doi: 10.1038/cdd.2016.156

    Figure Lengend Snippet: T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, ionomycin (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate

    Article Snippet: For ionomycin, LPS and IL-33 treatment survival assays, cells were washed to remove cytokines and then treated with 1 μ g/ml ionomycin, 5 μ g/ml ultrapure LPS ( E. coli 0111:B4 strain–TLR4 ligand, InvivoGen, San Diego, CA, USA), 10 ng/ml IL-33 (produced in-house and kindly provided by Dr. Ajithkumar Vasanthakumar of WEHI) or medium alone (no cytokines added).

    Techniques: Mouse Assay, FACS, Staining

    Siglec-9 blockade restores cytokine secretion and degranulation of NK cells in chronic hepatitis B (CHB) patients. PBMCs from CHB patients pretreated with Siglec-9 blocking antibody or isotype control IgG were stimulated with PMA plus ionomycin. Then, the expression of IFN-γ (A) , TNF-α (B) , and CD107a (C) were analyzed by flow cytometry. The right panels showed the representative flow cytometry data from one subject.

    Journal: Frontiers in Immunology

    Article Title: Decreased Siglec-9 Expression on Natural Killer Cell Subset Associated With Persistent HBV Replication

    doi: 10.3389/fimmu.2018.01124

    Figure Lengend Snippet: Siglec-9 blockade restores cytokine secretion and degranulation of NK cells in chronic hepatitis B (CHB) patients. PBMCs from CHB patients pretreated with Siglec-9 blocking antibody or isotype control IgG were stimulated with PMA plus ionomycin. Then, the expression of IFN-γ (A) , TNF-α (B) , and CD107a (C) were analyzed by flow cytometry. The right panels showed the representative flow cytometry data from one subject.

    Article Snippet: For Siglec-9 blockade assay, PBMCs were preincubated with human Siglec-9 antibody with a final concentration of 10 µg/ml or IgG controls (R & D, Minneapolis, MN, USA) for 40 min and then stimulated with PMA (50 ng/ml) (SIGMA, St. Louis, MO, USA), ionomycin (1 µg/ml) (BioLegend, San Diego, CA, USA), and Brefeldin A (10 µg/ml) (BioLegend, San Diego, CA, USA) for 4 h. After incubation with PerCp/cy5.5 anti-human CD3, PE/Cy7 anti-human CD56, cells were treated with the permeable agent Cytofix/Cytoperm (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions and were intracellularly stained with FITC anti-human IFN-γ (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) or APC anti-human TNF-α (eBioscience, San Diego, CA, USA) for 30 min.

    Techniques: Blocking Assay, Expressing, Flow Cytometry, Cytometry

    TAT-CBD3 inhibits the forward mode of NCX. Neurons were loaded with Fura-2FF. A , 5 μ m ionomycin in combination with 20 μ m AP-5 was applied to neurons. Where indicated, neurons were subjected to Na + /NMDG replacement ( B ) or treated with

    Journal: The Journal of Biological Chemistry

    Article Title: Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity *

    doi: 10.1074/jbc.M113.518472

    Figure Lengend Snippet: TAT-CBD3 inhibits the forward mode of NCX. Neurons were loaded with Fura-2FF. A , 5 μ m ionomycin in combination with 20 μ m AP-5 was applied to neurons. Where indicated, neurons were subjected to Na + /NMDG replacement ( B ) or treated with

    Article Snippet: Ionomycin was from LKT Laboratories (St. Paul, MN).

    Techniques:

    ATRA increases CD8+ T cell activation. (A) Representative flow cytometric gating strategy showing contour plots analyzing intracellular cytokine staining in unstimulated cells or cells stimulated for 5 hours with PMA + ionomycin. CD8(+) T cells were identified by first gating on single, live, CD3(+)CD8(+) cells. (B) Comparison of the pre-treatment and post-treatment frequency of CD8(+)IFNγ (+)CD107a(+) T cells. (C) Correlation between the frequencies of MDSCs and CD107a(+)IFNγ(+)CD8(+) T cells. Error bars indicate standard error of the mean. * Denotes p

    Journal: International immunopharmacology

    Article Title: Targeting myeloid-derived suppressor cells using all-trans retinoic acid in melanoma patients treated with Ipilimumab.

    doi: 10.1016/j.intimp.2018.08.007

    Figure Lengend Snippet: ATRA increases CD8+ T cell activation. (A) Representative flow cytometric gating strategy showing contour plots analyzing intracellular cytokine staining in unstimulated cells or cells stimulated for 5 hours with PMA + ionomycin. CD8(+) T cells were identified by first gating on single, live, CD3(+)CD8(+) cells. (B) Comparison of the pre-treatment and post-treatment frequency of CD8(+)IFNγ (+)CD107a(+) T cells. (C) Correlation between the frequencies of MDSCs and CD107a(+)IFNγ(+)CD8(+) T cells. Error bars indicate standard error of the mean. * Denotes p

    Article Snippet: Isolated PBMC were washed in phosphate buffered saline and incubated for 5 hours with FITC-anti-CD107a (Biolegend) and Protein Transport Inhibitor (eBioscience) in RPMI1640 as above with or without 50ng/mL phorbol 12-myristate 13-acetate (PMA), and 1μ g/mL ionomycin (Sigma-Aldrich).

    Techniques: Activation Assay, Flow Cytometry, Staining

    Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).

    Journal: Journal of Cell Science

    Article Title: Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

    doi: 10.1242/jcs.218958

    Figure Lengend Snippet: Specific F-actin structures in the sperm head did not change as a consequence of the AR initiation. Sperm were loaded with SiR-actin (green) and FM4-64 (magenta) and attached to concanavalin A-coated slides for imaging using TIRF microscopy. Following image acquisition, the ROIs indicated in A and D were analyzed. Representative image sequences of sperm stimulated with ionomycin (addition indicated by black arrow) that initially possessed the F-actin structure corresponding to the perforatorium (B), ventral (D; i) or neck (D; ii) region. The corresponding fluorescence traces (C and F) of the indicated ROIs (for SiR-actin) and the whole sperm head (for FM4-64) (A and D) are shown on the right. Analysis of these traces demonstrates that the depolymerization did not occur before or after the initiation of the AR (C and F) ( n =6. 62 cells analyzed).

    Article Snippet: Ionomycin was from Alomone Laboratories (Jerusalem, Israel).

    Techniques: Imaging, Microscopy, Fluorescence

    Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10†µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .

    Journal: Journal of Cell Science

    Article Title: Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis

    doi: 10.1242/jcs.218958

    Figure Lengend Snippet: Specific dynamic changes in the actin cytoskeleton after initiation of acrosomal exocytosis. (A) Sperm labeled with SiR-actin (green) and FM4-64 (magenta). The ROIs used for the analysis, corresponding to the upper acrosome region (for SiR-actin) and the whole sperm head (for FM4-64), are depicted. (B) Representative sequence of images of a sperm loaded with SiR-actin and FM4-64 that displayed a decrease in SiR-actin fluorescence in the upper acrosome region after stimulation with 10†µM ionomycin (addition indicated by black arrow). ″ represents time in seconds. (C) The resulting fluorescence traces revealed that the loss of F-actin occurred after the initiation of the AR as judged by the increase in FM4-64 fluorescence ( n .

    Article Snippet: Ionomycin was from Alomone Laboratories (Jerusalem, Israel).

    Techniques: Labeling, Sequencing, Fluorescence

    1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: 1-Methyl-tryptophan can interfere with TLR signaling in dendritic cells independently of IDO activity

    doi:

    Figure Lengend Snippet: 1-MT induces a Th2 function of DC stimulated with LPS. (A) Kinetic of secretion of IFNγ, IL-5 and IL-13 in MLR supernatants. MLR were conducted with control iDC (□), LPS-stimulated DC (○) and LPS-stimulated DC pre-treated with 1-MT (▲). Cytokines were measured in MLR supernatants at the indicated times. Mean ± SD of triplicates of one representative experiment out of three. (B) MLR were conducted for 5 days. After IL-2 expansion, T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A and IL-5, IL-13 and IFNγ expression was analyzed by intracellular staining. Data are shown for 1/20 DC/T cell ratio and were similar for other ratios. Data of one representative experiment out of three.

    Article Snippet: MLR were conducted for 5 days and T cells were expanded for 7 days with 25 U/ml rhIL-2 (Biosource), washed and restimulated with 10 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (VWR International, Fontenay-sous-Bois, France) for 5 h. 10 ng/ml Brefeldin A (Sigma-Aldrich) was added during the last 2 h. Cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences).

    Techniques: Expressing, Staining

    Quantification of amplitude of intercellular calcium signals and extent in cultured astrocytes. Analysis of intercellular calcium signaling generated ( A ) by mechanical stimulation ( n = 12) and ( B ) by focal application of ionomycin ( n = 21). Relative amplitude of [Ca 2+ ] i increases ( left scales , open columns ) and number of unresponsive cells ( right scales , dashed line ) were plotted by identifying the cellular row from which they were recorded. Cells were classified according to their location in reference to the stimulated cell, numbered 0 . Typically, stimulation was performed in the center of a microscopic field composed of ∼30 cells forming approximately six to seven cellular rows. As the distance from the stimulated cell increased, the amplitude of the response decreased, and more cells did not respond. For both modes of stimulation, the extent of intercellular calcium signaling generally exceeded the investigated cell population.

    Journal: The Journal of Neuroscience

    Article Title: Mechanism Involved in Initiation and Propagation of Receptor-Induced Intercellular Calcium Signaling in Cultured Rat Astrocytes

    doi: 10.1523/JNEUROSCI.17-06-01981.1997

    Figure Lengend Snippet: Quantification of amplitude of intercellular calcium signals and extent in cultured astrocytes. Analysis of intercellular calcium signaling generated ( A ) by mechanical stimulation ( n = 12) and ( B ) by focal application of ionomycin ( n = 21). Relative amplitude of [Ca 2+ ] i increases ( left scales , open columns ) and number of unresponsive cells ( right scales , dashed line ) were plotted by identifying the cellular row from which they were recorded. Cells were classified according to their location in reference to the stimulated cell, numbered 0 . Typically, stimulation was performed in the center of a microscopic field composed of ∼30 cells forming approximately six to seven cellular rows. As the distance from the stimulated cell increased, the amplitude of the response decreased, and more cells did not respond. For both modes of stimulation, the extent of intercellular calcium signaling generally exceeded the investigated cell population.

    Article Snippet: All drugs used were purchased from Sigma, except for U-73122 and U-73343 (Biomol, Plymouth, PA), ionomycin (Boehringer Mannheim, Mannheim, Germany), myo-[2-3 H]inositol (Amersham, Les Ulis, France), l (+)-2-amino-3-phosphonopropionic acid (L-AP3 ) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) (Tocris Cookson, Bristol, UK), and endothelin-1 (Neosystem, Strasbourg, France).

    Techniques: Cell Culture, Generated

    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Journal: Scientific Reports

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

    doi: 10.1038/s41598-018-29262-4

    Figure Lengend Snippet: CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Article Snippet: Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours.

    Techniques: Purification, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

    TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect, et al. Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect

    doi: 10.1111/cns.13062

    Figure Lengend Snippet: TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Article Snippet: Ionomycin was purchased from Merck.

    Techniques: Transfection, Western Blot, Cell Culture, Expressing

    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.

    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:

    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.

    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

    Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.

    Journal: JCI Insight

    Article Title: Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

    doi: 10.1172/jci.insight.98178

    Figure Lengend Snippet: Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.

    Article Snippet: Neutrophils were cultured at 37°C and 5% CO2 and stimulated with either 4 μM ionomycin (Adipogen) for 4 hours, 100 nM PMA (Sigma-Aldrich) for 4 hours, or MRSA (108 CFU/ml) for 20 minutes.

    Techniques: Mouse Assay, Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Immunofluorescence, In Vivo, Ex Vivo, Concentration Assay