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  • 95
    Thermo Fisher ionomycin
    Cytokine profiles of TFH and TFR cells from Peyer’s Patches (PP) and peripheral lymph node (pLN). Total cell preparations from PP and pLNs d6, d12, and d25 p.i. were in vitro stimulated with <t>PMA/ionomycin</t> in the presence of Brefeldin A, followed by intracellular detection of the indicated cytokines by flow cytometry. (A) Representative plots depicting expression of IL4, IL10, IL17, and IFNγ by TFH and TFR cells of PP or pLN 12 days p.i. as indicated are shown. (B) Summary of (A) (shown are mean ± SD). (C) 3D representation of time kinetics of cytokine production by pLN TFH and TFR cells from d6, d12, and d25 p.i. with KLH + Alum. Data from PP cells are also shown. Frequencies of positive cells are measured via intracellular detection of cytokines by flow cytometry as in (A) . Data were combined from two independent experiments with two mice per experiment for each time point. Shown are mean values.
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 4599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ionomycin
    Tbkbp1 is abundantly expressed in NKT cells and has a cell-intrinsic role in regulating NKT cell development. a qRT-PCR analyses of Tbkbp1 mRNA in total, CD4 + CD8 + double-positive (DP), CD4 – CD8 – double-negative (DN), and NKT thymic populations (upper) or splenic CD4 + and CD8 + T cells, NKT cells, and NK cells (lower). b IB analysis of Tbkbp1 and loading control Tubulin in the indicated splenic cell populations. c Flow cytometric analysis of NKT cell frequency and absolute numbers in the thymus (Thy), spleen (Spl) and liver (Liv) of age-matched WT and Tbkbp1 -KO (KO) mice, presented as representative plots (left) and summary graphs (right). n = 6 per genotype. d , e Flow cytometric analysis of NKT cell maturation stages (stage1: NK1.1 – CD44 – ; stage2: NK1.1 – CD44 + ; stage 3: NK1.1 + CD44 + ) in the thymus, spleen and liver of WT and Tbkbp1 -KO mice, presented as representative plots ( d ) and summary graphs ( e ). n = 6 per genotype. f , g Flow cytometric analysis of the indicated transcription factors in thymic ( f ) and splenic ( g ) NKT cells from WT and Tbkbp1 -KO mice, presented as representative plots (left) and summary graphs based on PLZF/RORγt flow values (right). n = 6 per genotype. h Flow cytometric analysis of thymic NKT cells based on IL-17Rb expression. i Flow cytometric analysis of IFNγ and IL-4 expression in WT and Tbkbp1 -KO thymic NKT cells after treatment for 4 h with PMA and <t>Ionomycin</t> in the presence of monensin, presented as representative plots and summary graphs ( n = 6 per genotype). j ELISA of IFN-γ and IL-4 in the supernatant of WT and Tbkbp1-KO thymic NKT cells after 48 h of in vitro stimulation with α-Galcer and antigen-presenting cells (WT BMDCs). k ELISA of IFNγ and IL-4 in the serum of WT and Tbkbp1- KO mice injected with α-Galcer (4 μg) for 6 h ( n = 5 per genotype). Data are representative of three independent experiments, and bar graphs are presented as mean ± s.d. values. * P
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ionomycin calcium salt
    NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and <t>ionomycin.</t> b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p
    Ionomycin Calcium Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cayman Chemical ionomycin
    NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and <t>ionomycin.</t> b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p
    Ionomycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 96/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pma ionomycin
    IL-12/23p40 + dendritic cells in spleen of WT and TNFRp55 -/- mice on ReA onset. Splenic cells were obtained from WT and TNFRp55 -/- mice on day 14 after Ye infection (ReA onset). Dendritic cells (DCs) were stained for cell surface CD11c and MHC-II, and analyzed by flow cytometry. (A) Absolute DC number in the spleen of Ye-infected WT and TNFRp55 -/- mice are presented. Splenic DCs of uninfected (PBS) mice were used as controls. (B) Representative dot plot showing analysis of IL-12/23p40 + DCs (CD11c + MHC-II + gate) in splenocytes from control (PBS), and Ye-infected WT and TNFRp55 -/- mice. The cells were stimulated with <t>PMA/Ionomycin</t> and brefeldin for 5 h, and then stained for cell surface CD11c and MHC-II, and intracellular IL-12/23p40, and then analyzed by flow cytometry. The numbers in the plots indicate the percentages of labeled cells in representative mice. (C) Percentage of IL-12/23p40 + DCs of the sum of three independent experiments. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. *** P
    Pma Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co ionomycin
    Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with <t>PMA/ionomycin</t> (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, 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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    99
    Millipore calcium ionophore ionomycin
    Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with <t>PMA/ionomycin</t> (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p
    Calcium Ionophore Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 185 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA ionomycin
    Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or <t>ionomycin</t> (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p
    Ionomycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam ionomycin
    Desialylation and apoptosis after cold storage. Platelet (PLT) desialylation (A and B) and apoptosis (C and D) were analyzed after seven days of storage, at room temperature (RT) or 4°C, in apheresis platelet concentrates (APCs) containing (A and C) 35% plasma (PAS-35-APC), (B and D) 20% plasma (PAS-20-APC) or 100% plasma (Plasma-APC). Desialylation was determined by measurement of FITC-labeled RCA (0.5 mg/mL) that binds to beta (β)-galactose using flow cytometetry. Fresh PLTs incubated with or without neuraminidase (Neu) were used as positive and negative control, respectively. The percentage of apoptotic cells was measured using FITC-labeled Annexin V. As positive (Pos ctr) and negative control (Neg ctr), freshly isolated PLTs incubated in the presence or in the absence of <t>Ionomycin</t> were used. Data are shown as mean±Standard Error of Mean of fluorescence intensity (MFI) and percentage of positive events, respectively. * P
    Ionomycin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    FUJIFILM ionomycin
    Ca 2+ signalling regulates Rac1 activity. ( a ) Rac1 activity after <t>Ionomycin</t> (Iono.) treatment for 3 minutes and 2 hours. DMZ explants were dissected and treated with Ionomycin. DMSO was used as a control. ( b ) Rac1 activity after Ionomycin treatment for 3 minutes. The active Rac1 intensity with Ionomycin was normalized to the intensity in DMSO-treated samples. Each western sample was prepared from 35–40 DMZ explants. Error bars indicate s.e. ± Student’s t-test, ns: No significance. ( c ) Rac1 activity after Ionomycin treatment for 2 hours. Student’s t-test, **P
    Ionomycin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant ionomycin
    IL-2Rα-KO Tregs acquire effector functionality. ( A ) Representative flow plots, and frequency of CD4 and CD8 Tregs in the spleen in 19-day-old WT and IL-2Rα-KO mice. n = 4–11 mice per experimental group. Representative flow plots and frequency of T cells producing IL-2 and IFNγ in 19-day-old WT and IL-2Rα-KO LN T cells ( B1 , B2 ) or Tregs ( C ) post 5-h PMA and <t>ionomycin</t> stimulation. n = 4–6 mice per experimental group. ( D ) CTLA-4 expression on CD4 Treg cells at day 19 by mean fluorescent intensity (MFI). ( E ) Frequency of IL-10+ 19-day-old WT and IL-2Rα-KO CD4 Treg cells post 5-h PMA and ionomycin stimulation. ( F ) Frequency of CTLA-4+FoxP3+CD4 T cells or CTLA-4+FoxP3− CD4 T cells are shown for 19-day-old WT and IL-2Rα-KO mice. ( G ) Frequency of IL-10+ 19-day-old WT and IL-2Rα-KO CD8 T and CD8 Treg cells post 5-h PMA and ionomycin stimulation. n = 4–6 mice per experimental group over 4 independent experiments. Statistics: unpaired Student’s t test; *p
    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
    Becton Dickinson pma ionomycin
    Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with <t>PMA+ionomycin.</t> In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P
    Pma Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific ionomycin
    Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM <t>ionomycin.</t> The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.
    Ionomycin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LC Laboratories ionomycin
    HDAC4 deficiency does not affect iNKT cell polarization A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic iNKT cells from HDAC4 WT and HDAC4 KO mice are shown. C ., D . Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of splenocytes with in vitro 4 hours PMA and <t>ionomycin</t> stimulation C . or in vivo 2 hours α-Galcer stimulation D . from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).
    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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    99
    Millipore ionomycin from streptomyces conglobatus
    HDAC4 deficiency does not affect iNKT cell polarization A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic iNKT cells from HDAC4 WT and HDAC4 KO mice are shown. C ., D . Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of splenocytes with in vitro 4 hours PMA and <t>ionomycin</t> stimulation C . or in vivo 2 hours α-Galcer stimulation D . from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).
    Ionomycin From Streptomyces Conglobatus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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: 96/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore ionomycin iono
    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 Iono, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Alomone Labs ionomycin
    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM <t>ionomycin</t> as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P
    Ionomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioVision ionomycin
    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM <t>ionomycin</t> as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P
    Ionomycin, supplied by BioVision, used in various techniques. Bioz Stars score: 94/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ionomycin free acid
    Membrane expansion occurs in parallel with conductance and current changes and requires the presence of monovalent cations. ( A ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were treated with 5 μM <t>ionomycin</t> for the period shown. Measurements were made of total capacitance, C m , transmembrane conductance, G m (lower blue trace), and transmembrane current I m (lower green trace). The upper red trace shows the change in capacitance (∆C m ), while the upper blue trace shows the integral ∫G m . Records were scaled to start and end at the same time, n = 10. ( B ) Membrane expansion was recorded as in ‘A’ and monovalent cation concentrations employed were varied as indicated.
    Ionomycin Free Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phorbol 12 myristate 13 acetate ionomycin
    Membrane expansion occurs in parallel with conductance and current changes and requires the presence of monovalent cations. ( A ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were treated with 5 μM <t>ionomycin</t> for the period shown. Measurements were made of total capacitance, C m , transmembrane conductance, G m (lower blue trace), and transmembrane current I m (lower green trace). The upper red trace shows the change in capacitance (∆C m ), while the upper blue trace shows the integral ∫G m . Records were scaled to start and end at the same time, n = 10. ( B ) Membrane expansion was recorded as in ‘A’ and monovalent cation concentrations employed were varied as indicated.
    Phorbol 12 Myristate 13 Acetate Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ionomycin is a selective calcium ionophore derived from S conglobatus that mobilizes intracellular calcium stores It is used as a research tool to raise the intracellular level of calcium to
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    Ionomycin free acid is an antibiotic selective calcium ionophore which can extract calcium ions from aqueous phase to organic phase In addition Ionomycin free acid acts as a transport carrier
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    Antibiotic that acts as a potent and selective calcium ionophore more effective than A23187 Ionomycin binds Ca2 in the 7 0 9 5 pH range with the resulting complex exhibiting
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    Image Search Results


    Cytokine profiles of TFH and TFR cells from Peyer’s Patches (PP) and peripheral lymph node (pLN). Total cell preparations from PP and pLNs d6, d12, and d25 p.i. were in vitro stimulated with PMA/ionomycin in the presence of Brefeldin A, followed by intracellular detection of the indicated cytokines by flow cytometry. (A) Representative plots depicting expression of IL4, IL10, IL17, and IFNγ by TFH and TFR cells of PP or pLN 12 days p.i. as indicated are shown. (B) Summary of (A) (shown are mean ± SD). (C) 3D representation of time kinetics of cytokine production by pLN TFH and TFR cells from d6, d12, and d25 p.i. with KLH + Alum. Data from PP cells are also shown. Frequencies of positive cells are measured via intracellular detection of cytokines by flow cytometry as in (A) . Data were combined from two independent experiments with two mice per experiment for each time point. Shown are mean values.

    Journal: Frontiers in Immunology

    Article Title: Shared and Unique Features Distinguishing Follicular T Helper and Regulatory Cells of Peripheral Lymph Node and Peyer’s Patches

    doi: 10.3389/fimmu.2018.00714

    Figure Lengend Snippet: Cytokine profiles of TFH and TFR cells from Peyer’s Patches (PP) and peripheral lymph node (pLN). Total cell preparations from PP and pLNs d6, d12, and d25 p.i. were in vitro stimulated with PMA/ionomycin in the presence of Brefeldin A, followed by intracellular detection of the indicated cytokines by flow cytometry. (A) Representative plots depicting expression of IL4, IL10, IL17, and IFNγ by TFH and TFR cells of PP or pLN 12 days p.i. as indicated are shown. (B) Summary of (A) (shown are mean ± SD). (C) 3D representation of time kinetics of cytokine production by pLN TFH and TFR cells from d6, d12, and d25 p.i. with KLH + Alum. Data from PP cells are also shown. Frequencies of positive cells are measured via intracellular detection of cytokines by flow cytometry as in (A) . Data were combined from two independent experiments with two mice per experiment for each time point. Shown are mean values.

    Article Snippet: Total cell preparations were plated at density of 5 × 106 /ml and incubated at 37°C for 4 h in RPMI 1640/10% FCS medium supplemented with 10 µM KN-62, Ionomycin 1.5 µg/ml (Invitrogen cat# I-24222), PMA 50 ng/ml (Calbiochem cat# 524400) and Brefeldin A 10 µg/ml (Sigma-Aldrich cat# B6542).

    Techniques: In Vitro, Flow Cytometry, Cytometry, Expressing, Mouse Assay

    Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p

    Journal: PLoS Pathogens

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    doi: 10.1371/journal.ppat.1006766

    Figure Lengend Snippet: Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p

    Article Snippet: R5421 was purchased from Sigma Aldrich (ANV00195), ionomycin (I24222) and staurosporine (PHZ1271) from Invitrogen Molecular Probes, and heparin sodium salt (H-3393) from Sigma Aldrich.

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

    Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    doi: 10.1371/journal.ppat.1006766

    Figure Lengend Snippet: Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.

    Article Snippet: R5421 was purchased from Sigma Aldrich (ANV00195), ionomycin (I24222) and staurosporine (PHZ1271) from Invitrogen Molecular Probes, and heparin sodium salt (H-3393) from Sigma Aldrich.

    Techniques: Staining, Fluorescence, Software, Incubation, Western Blot, Magnetic Beads, Immunoprecipitation

    Tbkbp1 is abundantly expressed in NKT cells and has a cell-intrinsic role in regulating NKT cell development. a qRT-PCR analyses of Tbkbp1 mRNA in total, CD4 + CD8 + double-positive (DP), CD4 – CD8 – double-negative (DN), and NKT thymic populations (upper) or splenic CD4 + and CD8 + T cells, NKT cells, and NK cells (lower). b IB analysis of Tbkbp1 and loading control Tubulin in the indicated splenic cell populations. c Flow cytometric analysis of NKT cell frequency and absolute numbers in the thymus (Thy), spleen (Spl) and liver (Liv) of age-matched WT and Tbkbp1 -KO (KO) mice, presented as representative plots (left) and summary graphs (right). n = 6 per genotype. d , e Flow cytometric analysis of NKT cell maturation stages (stage1: NK1.1 – CD44 – ; stage2: NK1.1 – CD44 + ; stage 3: NK1.1 + CD44 + ) in the thymus, spleen and liver of WT and Tbkbp1 -KO mice, presented as representative plots ( d ) and summary graphs ( e ). n = 6 per genotype. f , g Flow cytometric analysis of the indicated transcription factors in thymic ( f ) and splenic ( g ) NKT cells from WT and Tbkbp1 -KO mice, presented as representative plots (left) and summary graphs based on PLZF/RORγt flow values (right). n = 6 per genotype. h Flow cytometric analysis of thymic NKT cells based on IL-17Rb expression. i Flow cytometric analysis of IFNγ and IL-4 expression in WT and Tbkbp1 -KO thymic NKT cells after treatment for 4 h with PMA and Ionomycin in the presence of monensin, presented as representative plots and summary graphs ( n = 6 per genotype). j ELISA of IFN-γ and IL-4 in the supernatant of WT and Tbkbp1-KO thymic NKT cells after 48 h of in vitro stimulation with α-Galcer and antigen-presenting cells (WT BMDCs). k ELISA of IFNγ and IL-4 in the serum of WT and Tbkbp1- KO mice injected with α-Galcer (4 μg) for 6 h ( n = 5 per genotype). Data are representative of three independent experiments, and bar graphs are presented as mean ± s.d. values. * P

    Journal: Nature Communications

    Article Title: TBK-binding protein 1 regulates IL-15-induced autophagy and NKT cell survival

    doi: 10.1038/s41467-018-05097-5

    Figure Lengend Snippet: Tbkbp1 is abundantly expressed in NKT cells and has a cell-intrinsic role in regulating NKT cell development. a qRT-PCR analyses of Tbkbp1 mRNA in total, CD4 + CD8 + double-positive (DP), CD4 – CD8 – double-negative (DN), and NKT thymic populations (upper) or splenic CD4 + and CD8 + T cells, NKT cells, and NK cells (lower). b IB analysis of Tbkbp1 and loading control Tubulin in the indicated splenic cell populations. c Flow cytometric analysis of NKT cell frequency and absolute numbers in the thymus (Thy), spleen (Spl) and liver (Liv) of age-matched WT and Tbkbp1 -KO (KO) mice, presented as representative plots (left) and summary graphs (right). n = 6 per genotype. d , e Flow cytometric analysis of NKT cell maturation stages (stage1: NK1.1 – CD44 – ; stage2: NK1.1 – CD44 + ; stage 3: NK1.1 + CD44 + ) in the thymus, spleen and liver of WT and Tbkbp1 -KO mice, presented as representative plots ( d ) and summary graphs ( e ). n = 6 per genotype. f , g Flow cytometric analysis of the indicated transcription factors in thymic ( f ) and splenic ( g ) NKT cells from WT and Tbkbp1 -KO mice, presented as representative plots (left) and summary graphs based on PLZF/RORγt flow values (right). n = 6 per genotype. h Flow cytometric analysis of thymic NKT cells based on IL-17Rb expression. i Flow cytometric analysis of IFNγ and IL-4 expression in WT and Tbkbp1 -KO thymic NKT cells after treatment for 4 h with PMA and Ionomycin in the presence of monensin, presented as representative plots and summary graphs ( n = 6 per genotype). j ELISA of IFN-γ and IL-4 in the supernatant of WT and Tbkbp1-KO thymic NKT cells after 48 h of in vitro stimulation with α-Galcer and antigen-presenting cells (WT BMDCs). k ELISA of IFNγ and IL-4 in the serum of WT and Tbkbp1- KO mice injected with α-Galcer (4 μg) for 6 h ( n = 5 per genotype). Data are representative of three independent experiments, and bar graphs are presented as mean ± s.d. values. * P

    Article Snippet: PMA (P1585) and Ionomycin (I0634) were from Sigma-Aldrich.

    Techniques: Quantitative RT-PCR, Flow Cytometry, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Injection

    Tbkbp1 has a cell-intrinsic role in regulating NKT cell development. a , b Flow cytometric analysis of NKT cells in the thymus, spleen, and liver from 6-week-old WT and Tbkbp1 -TKO (TKO) mice, presented as a representative FACS plot of thymic NKT cells ( a ) and summary graphs of the indicated NKT cells (each circle represents a mouse) ( b ). c , d Flow cytometric analysis of thymic NKT cell maturation stages (stage 1: CD44 – NK1.1 – ; stage 2: CD44 + NK1.1 – ; stage 3: CD44 + NK1.1 + ), presented as a representative plot ( c ) and summary graphs ( d ). e Flow cytometric analysis of IL-4 expression in WT and Tbkbp1 -TKO thymic NKT cells after treatment for 4 h with PMA and ionomycin in the presence of monensin, presented as a representative plot and summary graph ( n = 5 per genotype). f , g Flow cytometric analysis of NKT cells and their maturation stages in the thymus ( f ) and spleen ( g ) of Rag1 -KO recipient mice adoptively transferred (for 6 week) with a mixture of BM cells derived from WT B6.SJL mice (CD45.1 + ) and Tbkbp1 -KO mice (CD45.2 + ), gating on CD45.1 + or CD45.2 + cells and presented as representative FACS plots and summary graphs ( n = 5 chimeric mice). h Flow cytometric analysis of cell surface expression of CD1d in DP thymocytes from WT or Tbkbp1-KO mice, presented as a representative FACS plot and summary graph ( n = 5 mice per genotype). i ELISA of IL-2 produced by NKT hybridoma cells cocultured for 24 h with total thymocytes from WT or Tbkbp1 -KO mice in the absence or presence of α-GalCer. Data are representative of at least three independent experiments, and bar graphs are presented as mean ± s.d. values. * P

    Journal: Nature Communications

    Article Title: TBK-binding protein 1 regulates IL-15-induced autophagy and NKT cell survival

    doi: 10.1038/s41467-018-05097-5

    Figure Lengend Snippet: Tbkbp1 has a cell-intrinsic role in regulating NKT cell development. a , b Flow cytometric analysis of NKT cells in the thymus, spleen, and liver from 6-week-old WT and Tbkbp1 -TKO (TKO) mice, presented as a representative FACS plot of thymic NKT cells ( a ) and summary graphs of the indicated NKT cells (each circle represents a mouse) ( b ). c , d Flow cytometric analysis of thymic NKT cell maturation stages (stage 1: CD44 – NK1.1 – ; stage 2: CD44 + NK1.1 – ; stage 3: CD44 + NK1.1 + ), presented as a representative plot ( c ) and summary graphs ( d ). e Flow cytometric analysis of IL-4 expression in WT and Tbkbp1 -TKO thymic NKT cells after treatment for 4 h with PMA and ionomycin in the presence of monensin, presented as a representative plot and summary graph ( n = 5 per genotype). f , g Flow cytometric analysis of NKT cells and their maturation stages in the thymus ( f ) and spleen ( g ) of Rag1 -KO recipient mice adoptively transferred (for 6 week) with a mixture of BM cells derived from WT B6.SJL mice (CD45.1 + ) and Tbkbp1 -KO mice (CD45.2 + ), gating on CD45.1 + or CD45.2 + cells and presented as representative FACS plots and summary graphs ( n = 5 chimeric mice). h Flow cytometric analysis of cell surface expression of CD1d in DP thymocytes from WT or Tbkbp1-KO mice, presented as a representative FACS plot and summary graph ( n = 5 mice per genotype). i ELISA of IL-2 produced by NKT hybridoma cells cocultured for 24 h with total thymocytes from WT or Tbkbp1 -KO mice in the absence or presence of α-GalCer. Data are representative of at least three independent experiments, and bar graphs are presented as mean ± s.d. values. * P

    Article Snippet: PMA (P1585) and Ionomycin (I0634) were from Sigma-Aldrich.

    Techniques: Flow Cytometry, Mouse Assay, FACS, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Produced

    NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and ionomycin. b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p

    Journal: Nature Communications

    Article Title: Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70

    doi: 10.1038/s41467-018-05095-7

    Figure Lengend Snippet: NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and ionomycin. b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p

    Article Snippet: For evaluation of IFN-γ production by iNKT cells directly ex vivo, freshly isolated PBMC and SFMC were incubated for 4 h with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml), calcium-ionomycin (CaI, 1 µg/ml; both from Sigma) and brefeldin A (BFA, 10 ng/ml; BD) or BFA alone (negative control).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Injection, Derivative Assay, Expressing

    IL-12/23p40 + dendritic cells in spleen of WT and TNFRp55 -/- mice on ReA onset. Splenic cells were obtained from WT and TNFRp55 -/- mice on day 14 after Ye infection (ReA onset). Dendritic cells (DCs) were stained for cell surface CD11c and MHC-II, and analyzed by flow cytometry. (A) Absolute DC number in the spleen of Ye-infected WT and TNFRp55 -/- mice are presented. Splenic DCs of uninfected (PBS) mice were used as controls. (B) Representative dot plot showing analysis of IL-12/23p40 + DCs (CD11c + MHC-II + gate) in splenocytes from control (PBS), and Ye-infected WT and TNFRp55 -/- mice. The cells were stimulated with PMA/Ionomycin and brefeldin for 5 h, and then stained for cell surface CD11c and MHC-II, and intracellular IL-12/23p40, and then analyzed by flow cytometry. The numbers in the plots indicate the percentages of labeled cells in representative mice. (C) Percentage of IL-12/23p40 + DCs of the sum of three independent experiments. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. *** P

    Journal: PLoS ONE

    Article Title: IL-12/23p40 overproduction by dendritic cells leads to an increased Th1 and Th17 polarization in a model of Yersinia enterocolitica-induced reactive arthritis in TNFRp55-/- mice

    doi: 10.1371/journal.pone.0193573

    Figure Lengend Snippet: IL-12/23p40 + dendritic cells in spleen of WT and TNFRp55 -/- mice on ReA onset. Splenic cells were obtained from WT and TNFRp55 -/- mice on day 14 after Ye infection (ReA onset). Dendritic cells (DCs) were stained for cell surface CD11c and MHC-II, and analyzed by flow cytometry. (A) Absolute DC number in the spleen of Ye-infected WT and TNFRp55 -/- mice are presented. Splenic DCs of uninfected (PBS) mice were used as controls. (B) Representative dot plot showing analysis of IL-12/23p40 + DCs (CD11c + MHC-II + gate) in splenocytes from control (PBS), and Ye-infected WT and TNFRp55 -/- mice. The cells were stimulated with PMA/Ionomycin and brefeldin for 5 h, and then stained for cell surface CD11c and MHC-II, and intracellular IL-12/23p40, and then analyzed by flow cytometry. The numbers in the plots indicate the percentages of labeled cells in representative mice. (C) Percentage of IL-12/23p40 + DCs of the sum of three independent experiments. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SEM. *** P

    Article Snippet: For intracellular staining of splenocytes, the cells were stimulated with PMA/ionomycin (50/750 ng/ml, eBioscience) for 5 h in the presence of GolgiPlug (BD Biosciences).

    Techniques: Mouse Assay, Infection, Staining, Flow Cytometry, Cytometry, Labeling

    Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p

    Journal: PLoS Pathogens

    Article Title: Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway

    doi: 10.1371/journal.ppat.1006819

    Figure Lengend Snippet: Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p

    Article Snippet: For intracellular cytokine detection, freshly isolated cells were stimulated for 6 h by culturing with PMA (50 ng/ml, Sigma) and ionomycin (1 μM, Merck) in the presence of BFA (1 μM).

    Techniques: Expressing

    Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p

    Journal: PLoS Pathogens

    Article Title: Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway

    doi: 10.1371/journal.ppat.1006819

    Figure Lengend Snippet: Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p

    Article Snippet: For intracellular cytokine detection, freshly isolated cells were stimulated for 6 h by culturing with PMA (50 ng/ml, Sigma) and ionomycin (1 μM, Merck) in the presence of BFA (1 μM).

    Techniques: Infection

    Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Ca 2+ influx via voltage-gated calcium channels is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM flunarizine (Flun) for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 5 for both P-Akt and P-GSK3 with 80 Hz, n = 6 for P-Akt IONO and n = 7 for P-GSK3 IONO (students t test, ns non-significant, * p

    Article Snippet: EGTA-AM was from Cambridge Bioscience and ionomycin from Merck Chemicals.

    Techniques: Incubation

    Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Localised Ca 2+ influx is essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with either 100 μM BAPTA-AM or EGTA-AM for 30 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 4 for P-Akt 80 Hz, n = 5 for P-GSK3 80 Hz, n = 7 for P-Akt IONO and n = 6 for P-GSK3 IONO (students t test, ns non-significant * p

    Article Snippet: EGTA-AM was from Cambridge Bioscience and ionomycin from Merck Chemicals.

    Techniques: Incubation

    [Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: [Ca 2+ ] i increases are essential for Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then either rested (Basal) or stimulated with ionomycin (IONO, 5 µM) for 1 min in incubation buffer containing either 1.3 mM (+Ca 2+ ) or low (−Ca 2+ ) calcium. a Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ). b , c The fold increase in phosphorylation of either Akt Ser473 ( b , open bars ) or GSK3α/β Ser21/9 ( c , closed bars ) is displayed after correction for protein levels using β-Actin and normalisation to the low calcium control. All error bars represent ±SEM; n = 5 for P-Akt and n = 4 for P-GSK3 (students t test, ns non-significant, * p

    Article Snippet: EGTA-AM was from Cambridge Bioscience and ionomycin from Merck Chemicals.

    Techniques: Incubation

    Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p

    Journal: Neurochemical Research

    Article Title: Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals

    doi: 10.1007/s11064-015-1663-5

    Figure Lengend Snippet: Calmodulin is not the calcium sensor for activity-dependent Akt/GSK3 phosphorylation. CGNs were removed from culture medium and repolarised in incubation medium for 10 min. Cultures were then incubated with or without incubation medium supplemented with 10 μM calmidazolium for 10 min. After this point CGNs were left to rest or challenged with either 800 action potentials (80 Hz) or ionomycin (IONO, 2.5 µM for 1 min). a , d Representative blots are displayed showing either Akt Ser473 phosphorylation ( P - Akt ), GSK3α/β Ser21/9 phosphorylation ( P - GSK3 ) or β-Actin levels ( β - Actin ) after either action potential ( a ) or ionomycin ( d ) stimulation. b , e The fold increase in phosphorylation of Akt Ser473 ( open bars ) in response to either action potentials ( b ) or ionomycin ( e ) is displayed. c , f The fold increase in phosphorylation of GSK3α/β Ser21/9 ( closed bars ) in response to either action potentials ( c ) or ionomycin ( f ) is displayed. In all cases phosphorylation levels were corrected for protein levels using β-Actin and normalisation to the basal controls. All error bars represent ±SEM; n = 6 for P-Akt 80 Hz, n = 9 for P-GSK3 80 Hz, n = 8 for P-Akt IONO and n = 9 for P-GSK3 IONO (students t test, ns non-significant, * p

    Article Snippet: EGTA-AM was from Cambridge Bioscience and ionomycin from Merck Chemicals.

    Techniques: Activity Assay, Incubation

    Desialylation and apoptosis after cold storage. Platelet (PLT) desialylation (A and B) and apoptosis (C and D) were analyzed after seven days of storage, at room temperature (RT) or 4°C, in apheresis platelet concentrates (APCs) containing (A and C) 35% plasma (PAS-35-APC), (B and D) 20% plasma (PAS-20-APC) or 100% plasma (Plasma-APC). Desialylation was determined by measurement of FITC-labeled RCA (0.5 mg/mL) that binds to beta (β)-galactose using flow cytometetry. Fresh PLTs incubated with or without neuraminidase (Neu) were used as positive and negative control, respectively. The percentage of apoptotic cells was measured using FITC-labeled Annexin V. As positive (Pos ctr) and negative control (Neg ctr), freshly isolated PLTs incubated in the presence or in the absence of Ionomycin were used. Data are shown as mean±Standard Error of Mean of fluorescence intensity (MFI) and percentage of positive events, respectively. * P

    Journal: Haematologica

    Article Title: Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates

    doi: 10.3324/haematol.2018.195057

    Figure Lengend Snippet: Desialylation and apoptosis after cold storage. Platelet (PLT) desialylation (A and B) and apoptosis (C and D) were analyzed after seven days of storage, at room temperature (RT) or 4°C, in apheresis platelet concentrates (APCs) containing (A and C) 35% plasma (PAS-35-APC), (B and D) 20% plasma (PAS-20-APC) or 100% plasma (Plasma-APC). Desialylation was determined by measurement of FITC-labeled RCA (0.5 mg/mL) that binds to beta (β)-galactose using flow cytometetry. Fresh PLTs incubated with or without neuraminidase (Neu) were used as positive and negative control, respectively. The percentage of apoptotic cells was measured using FITC-labeled Annexin V. As positive (Pos ctr) and negative control (Neg ctr), freshly isolated PLTs incubated in the presence or in the absence of Ionomycin were used. Data are shown as mean±Standard Error of Mean of fluorescence intensity (MFI) and percentage of positive events, respectively. * P

    Article Snippet: Freshly isolated PLTs were incubated with 10 mM ionomycin (Abcam, Cambridge, UK) and used as positive control.

    Techniques: Labeling, Flow Cytometry, Incubation, Negative Control, Isolation, Fluorescence

    Ca 2+ signalling regulates Rac1 activity. ( a ) Rac1 activity after Ionomycin (Iono.) treatment for 3 minutes and 2 hours. DMZ explants were dissected and treated with Ionomycin. DMSO was used as a control. ( b ) Rac1 activity after Ionomycin treatment for 3 minutes. The active Rac1 intensity with Ionomycin was normalized to the intensity in DMSO-treated samples. Each western sample was prepared from 35–40 DMZ explants. Error bars indicate s.e. ± Student’s t-test, ns: No significance. ( c ) Rac1 activity after Ionomycin treatment for 2 hours. Student’s t-test, **P

    Journal: Scientific Reports

    Article Title: Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation

    doi: 10.1038/s41598-018-20747-w

    Figure Lengend Snippet: Ca 2+ signalling regulates Rac1 activity. ( a ) Rac1 activity after Ionomycin (Iono.) treatment for 3 minutes and 2 hours. DMZ explants were dissected and treated with Ionomycin. DMSO was used as a control. ( b ) Rac1 activity after Ionomycin treatment for 3 minutes. The active Rac1 intensity with Ionomycin was normalized to the intensity in DMSO-treated samples. Each western sample was prepared from 35–40 DMZ explants. Error bars indicate s.e. ± Student’s t-test, ns: No significance. ( c ) Rac1 activity after Ionomycin treatment for 2 hours. Student’s t-test, **P

    Article Snippet: To treat whole embryos, Ionomycin (095–05831, Wako) in DMSO was added to the medium at st. 11.

    Techniques: Activity Assay, Western Blot

    Intracellular Ca 2+ signalling regulates migration activity in the LEM. ( a ) Snapshots from time-lapse imaging of the Ca 2+ dynamics in DMZ explants treated with Ionomycin (2.5 μM). Upper panel: mRFP. Lower panel: FRET ratio of yellow cameleon-nano converted to pseudocolours (bar at right). ( b ) LEM migration into open space in Ionomycin- (2.5 μM) and DMSO-treated DMZ explants monitored by mRFP. ( c ) Relative migration distance in DMSO- and Ionomycin-treated DMZ explants. The migration distance was normalized to the migration distance of DMSO-treated DMZ. DMSO: n = 7 embryos. Ionomycin (Iono.): n = 8 embryos. Error bars indicate s.e. ± Student’s t-test, *P

    Journal: Scientific Reports

    Article Title: Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation

    doi: 10.1038/s41598-018-20747-w

    Figure Lengend Snippet: Intracellular Ca 2+ signalling regulates migration activity in the LEM. ( a ) Snapshots from time-lapse imaging of the Ca 2+ dynamics in DMZ explants treated with Ionomycin (2.5 μM). Upper panel: mRFP. Lower panel: FRET ratio of yellow cameleon-nano converted to pseudocolours (bar at right). ( b ) LEM migration into open space in Ionomycin- (2.5 μM) and DMSO-treated DMZ explants monitored by mRFP. ( c ) Relative migration distance in DMSO- and Ionomycin-treated DMZ explants. The migration distance was normalized to the migration distance of DMSO-treated DMZ. DMSO: n = 7 embryos. Ionomycin (Iono.): n = 8 embryos. Error bars indicate s.e. ± Student’s t-test, *P

    Article Snippet: To treat whole embryos, Ionomycin (095–05831, Wako) in DMSO was added to the medium at st. 11.

    Techniques: Migration, Activity Assay, Imaging

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-78975-y

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

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

    Techniques: Mouse Assay, Expressing

    Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with PMA+ionomycin. In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Analysis of soluble cytokines after polyclonal stimulation or stimulation with an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of soluble cytokines (pg/ml) among age groups younger and older than 50 years of age after stimulation with PMA+ionomycin. In control samples, only dimethyl sulphoxide and ethanol were used. (b) Comparative analysis of soluble cytokines among age groups after stimulation with EBV lysate. In control samples, only the co-stimulating molecules anti-CD49 and anti-CD28 were used. The boxes represent the median and range data. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques:

    Percentage of cytokine-producing T cells after short-term stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of the frequencies of monofunctional and multi-functional CD4 + and CD8 + T lymphocytes for tumour necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ after stimulation with PMA+ionomycin among individuals younger and older than 50 years of age. (b) Comparative analysis of T lymphocyte functionality between age groups after stimulation with EBV lysate. The middle line and the vertical lines represent the median and ranges, respectively. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Percentage of cytokine-producing T cells after short-term stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Comparative analysis of the frequencies of monofunctional and multi-functional CD4 + and CD8 + T lymphocytes for tumour necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ after stimulation with PMA+ionomycin among individuals younger and older than 50 years of age. (b) Comparative analysis of T lymphocyte functionality between age groups after stimulation with EBV lysate. The middle line and the vertical lines represent the median and ranges, respectively. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques: Functional Assay

    Frequency of tumour necrosis factor (TNF)-α + T cells after in-vitro stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Frequency of CD4 + and CD8 + T cells (TNF-α + ) after polyclonal stimulation with PMA+ionomycin in the peripheral blood (PB) of healthy subjects grouped according to age. (b) Relative frequencies of activated CD4 + and CD8 + T cells (TNF-α + ) in the absence and presence of EBV lysate in individuals from the two age groups. (c) Comparison of the specific T cell response after stimulus with EBV lysate among healthy individuals. In all figures, the median and range are shown. * P

    Journal: Clinical and Experimental Immunology

    Article Title: Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

    doi: 10.1111/cei.12337

    Figure Lengend Snippet: Frequency of tumour necrosis factor (TNF)-α + T cells after in-vitro stimulation with phorbol myristate acetate (PMA)+ionomycin or an Epstein–Barr virus (EBV) lysate. (a) Frequency of CD4 + and CD8 + T cells (TNF-α + ) after polyclonal stimulation with PMA+ionomycin in the peripheral blood (PB) of healthy subjects grouped according to age. (b) Relative frequencies of activated CD4 + and CD8 + T cells (TNF-α + ) in the absence and presence of EBV lysate in individuals from the two age groups. (c) Comparison of the specific T cell response after stimulus with EBV lysate among healthy individuals. In all figures, the median and range are shown. * P

    Article Snippet: Soluble cytokine levels were measured in the culture supernatants of in-vitro -cultured PB samples after short-term (6 h) stimulation with EBV or PMA+ionomycin or no stimulation, using the BD cytometric bead array (CBA) human T helper type 1 (Th1)/Th2 cytokine kit II according to the manufacturer's recommendations.

    Techniques: In Vitro

    Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    HDAC4 deficiency does not affect iNKT cell polarization A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic iNKT cells from HDAC4 WT and HDAC4 KO mice are shown. C ., D . Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of splenocytes with in vitro 4 hours PMA and ionomycin stimulation C . or in vivo 2 hours α-Galcer stimulation D . from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).

    Journal: Oncotarget

    Article Title: HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function

    doi: 10.18632/oncotarget.15077

    Figure Lengend Snippet: HDAC4 deficiency does not affect iNKT cell polarization A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic iNKT cells from HDAC4 WT and HDAC4 KO mice are shown. C ., D . Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of splenocytes with in vitro 4 hours PMA and ionomycin stimulation C . or in vivo 2 hours α-Galcer stimulation D . from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).

    Article Snippet: Cell stimulation in vitro and intracellular cytokine assays Total splenocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS and stimulated with PMA (50ng/ml, LC laboratory) plus Ionomycin (747ng/ml, LC laboratory).

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, In Vitro, In Vivo

    Unaltered Th1, Th2 and Th17 cell differentiation in HDAC4-deficient mice A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic conventional CD4 + T cells from HDAC4 WT and HDAC4 KO mice are shown. B . Whole splenocytes from HDAC4 WT and HDAC4 KO mice were treated with PMA and ionomycin in vitro for 4 h. Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of CD4 + T cells from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).

    Journal: Oncotarget

    Article Title: HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function

    doi: 10.18632/oncotarget.15077

    Figure Lengend Snippet: Unaltered Th1, Th2 and Th17 cell differentiation in HDAC4-deficient mice A . Flow cytometry analysis (left panel), histogram (middle panel) and frequencies (right panel) of T-bet, GATA3 and RORγt expressions in splenic conventional CD4 + T cells from HDAC4 WT and HDAC4 KO mice are shown. B . Whole splenocytes from HDAC4 WT and HDAC4 KO mice were treated with PMA and ionomycin in vitro for 4 h. Flow cytometry analysis (left panel) and frequencies (right panel) of CD69, IL4, IFN-γ, IL17 and TNF-α expression of CD4 + T cells from HDAC4 WT and HDAC4 KO mice are shown. Data represents three independent experiments with 2 to 3 mice per experiment (mean ± SD).

    Article Snippet: Cell stimulation in vitro and intracellular cytokine assays Total splenocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FCS and stimulated with PMA (50ng/ml, LC laboratory) plus Ionomycin (747ng/ml, LC laboratory).

    Techniques: Cell Differentiation, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, 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

    Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Directionality of the [Ca 2+ ]i increase wave propagation in response to progesterone in mouse sperm immobilized with concanavalin A. A ) Representative diagram of the different sperm areas evaluated in sperm attached to concanavalin A-coated slides. PPP, posterior principal piece; PPP II , posterior principal piece II; PPP I , posterior principal piece I; APP, anterior principal piece; CD, cytoplasmic droplet; PMP, posterior midpiece; AMP, anterior midpiece; H, head. B and E ) Representative images of the time course of Fluo-4 AM-labeled sperm after addition of 100 μM progesterone or 10 μM ionomycin as a control, respectively. C and F ) Time course of the fluorescence changes in each of the areas of the sperm shown in B and E , respectively. D and G ) Different time delays of the analyzed areas of the [Ca 2+ ]i increase propagation in sperm stimulated with progesterone or ionomycin, respectively (n = 8). Statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Labeling, Fluorescence

    [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: [Ca 2+ ]i and AR alternate measurements in mouse sperm. A and B ) Fluo-4 AM and FM4-64 fluorescence from sperm exposed to progesterone (PROG) or ionomycin (IONO), respectively. The arrow in the panel corresponding to 75 sec indicates a sperm that increased its [Ca 2+ ]i and underwent AR (arrow in panel corresponding to 350 sec). C ) Representative alternate Fluo-4 AM and FM4-64 fluorescence images of a sperm undergoing a progesterone response. C ) Fluo-4 AM and FM4-64 fluorescence changes observed in the sperm displayed in D . E ) Alternate Fluo-4 AM and FM4-64 fluorescence images of a representative sperm responding to ionomycin. F ) Fluo-4 AM and FM4-64 fluorescence changes observed during the recordings of the sperm shown in E . G ) Percentage of AR stimulated by progesterone or ionomycin at different times (AR time was established as the time that FM4-64 fluorescence significantly increased); n = 8 mice, 21 reacted cells and n = 4 mice, 43 reacted cells for progesterone and ionomycin experiments, respectively. Statistically significant differences between the progesterone and ionomycin response at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Fluorescence, Size-exclusion Chromatography, Mouse Assay

    Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Validation of the plasma membrane probe FM4-64 as an AR indicator and its simultaneous observation with the calcium indicator Fluo-4 AM. A ) Representative fluorescence images of the time course of an FM4-64-labeled EGFP-sperm that does not undergo AR after addition of 10 μM ionomycin (IONO). B ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in A . C ) Representative fluorescence images of the time course of FM4-64-labeled EGFP-sperm undergoing AR after addition of 10 μM ionomycin. D ) EGFP and FM4-64 fluorescence intensities (in arbitrary units) of the sperm shown in C . E ) Fluorescence images of FM4-64- and Fluo-4 AM-labeled sperm.

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Fluorescence, Labeling

    Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Mouse sperm displayed different patterns of [Ca 2+ ]i increase in response to progesterone. A ) Graphics representing the different patterns of [Ca 2+ ]i increase observed in the sperm head as a result of the addition of 100 μM progesterone (PROG). According to the calcium response, the patterns of increase were classified as sustained, transitory, oscillatory, late transitory, and gradual increase. The bar below the traces represents the time scale = 1 min. B ) Representative time lapse images of Fluo-4 AM-loaded sperm following the addition of 100 μM progesterone and 10 μM ionomycin (IONO). C ) Percentage of sperm displaying each of the patterns observed. These results were obtained by analyzing 184 sperm from 10 mice. D ) Comparison of the calcium increase in response to progesterone normalized by the ionomycin response. These results were obtained by analyzing 184 sperm from 10 mice; statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Mouse Assay

    Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Journal: Biology of Reproduction

    Article Title: A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm 1

    doi: 10.1095/biolreprod.115.136085

    Figure Lengend Snippet: Progesterone promotes an [Ca 2+ ]i increase in Fluo-4 AM-loaded mice sperm. A ) Image sequence showing sperm exposed to 40 μM progesterone (PROG) and recorded for 30 min. Sperm displayed an [Ca 2+ ]i increase at different times during the experiment. At the end of the recording, ionomycin (IONO) was added as a viability control. B ) Fluorescence changes corresponding to sperm 1–3 of the field recorded in A . C ) Percentage of sperm displaying an [Ca 2+ ]i increase in response to vehicle, 40 μM, and 100 μM progesterone addition. Black arrows indicate the time where progesterone and ionomycin were applied. These results were obtained by analyzing 319 sperm from 25 mice; statistically significant differences at * P

    Article Snippet: Bovine serum albumin, concanavalin A, and progesterone were purchased from Sigma-Aldrich Chemical Co. Ionomycin was from Alomone Labs.

    Techniques: Mouse Assay, Sequencing, Fluorescence

    Membrane expansion occurs in parallel with conductance and current changes and requires the presence of monovalent cations. ( A ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , transmembrane conductance, G m (lower blue trace), and transmembrane current I m (lower green trace). The upper red trace shows the change in capacitance (∆C m ), while the upper blue trace shows the integral ∫G m . Records were scaled to start and end at the same time, n = 10. ( B ) Membrane expansion was recorded as in ‘A’ and monovalent cation concentrations employed were varied as indicated.

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Membrane expansion occurs in parallel with conductance and current changes and requires the presence of monovalent cations. ( A ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , transmembrane conductance, G m (lower blue trace), and transmembrane current I m (lower green trace). The upper red trace shows the change in capacitance (∆C m ), while the upper blue trace shows the integral ∫G m . Records were scaled to start and end at the same time, n = 10. ( B ) Membrane expansion was recorded as in ‘A’ and monovalent cation concentrations employed were varied as indicated.

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Incubation

    Plasma membrane internalization in TMEM16F-null Jurkat T cells treated with ionomycin. ( A ) TMEM16F-null Jurkat T cells were incubated with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 15 minutes at 37 °C. Cells were then chilled on ice and stained with Annexin V (Anx V) to detect surface phosphatidylserine. Flow cytometry analysis shows intracellular calcium plotted against surface phosphatidylserine. ( B ) Single TMEM16F-null Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were then treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , which reflects plasma membrane area, and transmembrane conductance, G m . The red trace shows the change in capacitance (∆C m ) compared to t = 0, while the blue trace shows G m . A typical ∆C m trace is shown, the error bar represents standard error of the mean (SEM) at 150 s post ionomycin addition (n = 10). In addition, the dye FM4-64, which binds reversibly to membranes, was added to the same patched cell and removed as shown. The solid trace shows total FM4-64 fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of FM4-64 fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm).

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Plasma membrane internalization in TMEM16F-null Jurkat T cells treated with ionomycin. ( A ) TMEM16F-null Jurkat T cells were incubated with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 15 minutes at 37 °C. Cells were then chilled on ice and stained with Annexin V (Anx V) to detect surface phosphatidylserine. Flow cytometry analysis shows intracellular calcium plotted against surface phosphatidylserine. ( B ) Single TMEM16F-null Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were then treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , which reflects plasma membrane area, and transmembrane conductance, G m . The red trace shows the change in capacitance (∆C m ) compared to t = 0, while the blue trace shows G m . A typical ∆C m trace is shown, the error bar represents standard error of the mean (SEM) at 150 s post ionomycin addition (n = 10). In addition, the dye FM4-64, which binds reversibly to membranes, was added to the same patched cell and removed as shown. The solid trace shows total FM4-64 fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of FM4-64 fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm).

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Confocal Microscopy

    TMEM16F-null Jurkat T cells internalise PD-1 upon treatment with ionomycin ( A ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. ( B ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( C ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( D ) TMEM16F-null Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). Cellular FACS data shows the mean +/− SEM for three independent experiments. (****p

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: TMEM16F-null Jurkat T cells internalise PD-1 upon treatment with ionomycin ( A ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. ( B ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( C ) TMEM16F-null Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( D ) TMEM16F-null Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). Cellular FACS data shows the mean +/− SEM for three independent experiments. (****p

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Expressing, Staining, FACS, Sequencing, Microscopy

    Simultaneous surface phosphatidylserine exposure and plasma membrane expansion is followed by membrane vesicle shedding. ( A ) Jurkat T cells were loaded with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 400 s at 37 °C in the presence of polylysine-rhodamine (K7r) which binds rapidly to exposed phosphatidylserine. Confocal microscope images of Fluo4-AM and K7r fluorescence in a single field of cells are shown (scale bar is 10 μm). ( B ) A single TMEM16F-null Jurkat T cells was patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods), incubated at 37 °C in Ringer’s solution, then treated with 5 μM ionomycin at the time shown. Total capacitance, C m , was measured and the red trace shows the change in capacitance (∆C m ) compared to t = 0. In addition, K7r was added to the same patched cell and removed as shown. The green trace shows total K7r fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of K7r fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm). (C) Wild-type or TMEM16F-null Jurkat T cells were treated with 5 μM ionomycin for 15 minutes in the presence of eFluor780, a plasma membrane impermeable dye. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized particles using forward scatter and side scatter. Staining of these subcellular particles is displayed versus side scatter, with unstained events representing dye impermeable membrane vesicles rather than apoptotic bodies. The numbers shown represent the percentage of total events.

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Simultaneous surface phosphatidylserine exposure and plasma membrane expansion is followed by membrane vesicle shedding. ( A ) Jurkat T cells were loaded with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 400 s at 37 °C in the presence of polylysine-rhodamine (K7r) which binds rapidly to exposed phosphatidylserine. Confocal microscope images of Fluo4-AM and K7r fluorescence in a single field of cells are shown (scale bar is 10 μm). ( B ) A single TMEM16F-null Jurkat T cells was patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods), incubated at 37 °C in Ringer’s solution, then treated with 5 μM ionomycin at the time shown. Total capacitance, C m , was measured and the red trace shows the change in capacitance (∆C m ) compared to t = 0. In addition, K7r was added to the same patched cell and removed as shown. The green trace shows total K7r fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images of K7r fluorescence taken using confocal microscopy at the time points shown (scale bar is 5 μm). (C) Wild-type or TMEM16F-null Jurkat T cells were treated with 5 μM ionomycin for 15 minutes in the presence of eFluor780, a plasma membrane impermeable dye. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized particles using forward scatter and side scatter. Staining of these subcellular particles is displayed versus side scatter, with unstained events representing dye impermeable membrane vesicles rather than apoptotic bodies. The numbers shown represent the percentage of total events.

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Microscopy, Fluorescence, Incubation, Confocal Microscopy, FACS, Staining

    Plasma membrane expansion activity of TMEM16F mutants ( A ) Single HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; transient increase of extracellular [Ca 2+ ] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca 2+ . The change in membrane capacitance (∆C m ) was measured and the percentage change was calculated. Single recordings are displayed, mean values at 30 s, 45 s and 60 s after stimulation are shown (+/−SEM, n = 7). Upper red trace, control cells; lower blue trace, cells over-expressing murine TMEM16F. ( B ) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; transient increase of extracellular [Ca 2+ ] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca 2+ . The change in membrane capacitance (∆C m ) was measured and the percentage change was calculated. Single recordings are displayed with mean values at 30 s, 45 s and 60 s after stimulation shown (+/−SEM, n = 7). Upper red trace, control cells; lower traces, cells over-expressing murine TMEM16F and mutants as shown. ( C ) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; a transient increase of extracellular [Ca 2+ ] to 2 mM was used to induce an increase in intracellular Ca 2+ in the presence of the phosphatidylserine binding dye K7r. In the case of “WT rescue (iono)” cells were treated with 5 μM ionomycin instead. The total fluorescence of each cell was measured at 60 s using a confocal microscope, and the percentage change compared to untreated was calculated. Mean values are shown (+/−SEM, n = 7; **p

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Plasma membrane expansion activity of TMEM16F mutants ( A ) Single HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; transient increase of extracellular [Ca 2+ ] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca 2+ . The change in membrane capacitance (∆C m ) was measured and the percentage change was calculated. Single recordings are displayed, mean values at 30 s, 45 s and 60 s after stimulation are shown (+/−SEM, n = 7). Upper red trace, control cells; lower blue trace, cells over-expressing murine TMEM16F. ( B ) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; transient increase of extracellular [Ca 2+ ] to 2 mM (at the time shown) was used to induce an increase in intracellular Ca 2+ . The change in membrane capacitance (∆C m ) was measured and the percentage change was calculated. Single recordings are displayed with mean values at 30 s, 45 s and 60 s after stimulation shown (+/−SEM, n = 7). Upper red trace, control cells; lower traces, cells over-expressing murine TMEM16F and mutants as shown. ( C ) Single TMEM16F-null HEK293 cells stably expressing the cardiac Na/Ca exchanger (NCX1.1) were patched with a glass micropipette loaded with 40 mM Na + ; a transient increase of extracellular [Ca 2+ ] to 2 mM was used to induce an increase in intracellular Ca 2+ in the presence of the phosphatidylserine binding dye K7r. In the case of “WT rescue (iono)” cells were treated with 5 μM ionomycin instead. The total fluorescence of each cell was measured at 60 s using a confocal microscope, and the percentage change compared to untreated was calculated. Mean values are shown (+/−SEM, n = 7; **p

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Activity Assay, Stable Transfection, Expressing, Binding Assay, Fluorescence, Microscopy

    Surface phosphatidylserine exposure and plasma membrane expansion in Jurkat T cells treated with ionomycin. ( A ) Jurkat T cells were incubated with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 15 minutes at 37 °C. Cells were then chilled on ice and stained with Annexin V (Anx V) to detect surface phosphatidylserine. Flow cytometry analysis shows intracellular calcium plotted against surface phosphatidylserine. ( B ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were then treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , which reflects plasma membrane area, and transmembrane conductance, G m . The red trace shows the change in capacitance (∆C m ) compared to t = 0, while the blue trace shows G m . A typical ∆C m trace is shown, the error bar represents standard error of the mean (SEM) at 150 s post ionomycin addition (n = 10). In addition, the dye FM4-64, which binds reversibly to membranes, was added to the same patched cell and removed as shown. The solid trace shows total FM4-64 fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images taken using confocal microscopy at the time points shown (scale bar is 5 μm).

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Surface phosphatidylserine exposure and plasma membrane expansion in Jurkat T cells treated with ionomycin. ( A ) Jurkat T cells were incubated with the cytoplasmic calcium indicator Fluo4-AM, then treated with 5 μM ionomycin for 15 minutes at 37 °C. Cells were then chilled on ice and stained with Annexin V (Anx V) to detect surface phosphatidylserine. Flow cytometry analysis shows intracellular calcium plotted against surface phosphatidylserine. ( B ) Single Jurkat T cells were patched with a glass micropipette loaded with cytoplasmic solution (see Materials and Methods) and incubated at 37 °C in Ringer’s solution. Cells were then treated with 5 μM ionomycin for the period shown. Measurements were made of total capacitance, C m , which reflects plasma membrane area, and transmembrane conductance, G m . The red trace shows the change in capacitance (∆C m ) compared to t = 0, while the blue trace shows G m . A typical ∆C m trace is shown, the error bar represents standard error of the mean (SEM) at 150 s post ionomycin addition (n = 10). In addition, the dye FM4-64, which binds reversibly to membranes, was added to the same patched cell and removed as shown. The solid trace shows total FM4-64 fluorescence of the cell, measured by a confocal microscope, relative to t = 0. Below are images taken using confocal microscopy at the time points shown (scale bar is 5 μm).

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Confocal Microscopy

    Wild-type Jurkat T cells shed PD-1 upon treatment with ionomycin ( A ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. ( B ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( C ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (D) Control wild-type Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). ( E ) Control wild-type Jurkat T cells, or control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, were stained with an anti-PD-1 antibody, then treated with 5 μM ionomycin for 15 minutes at 37 °C in the presence of fixable viability dye eFluor780. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized events (low forward scatter) and intact membranes (low fixable viability dye eFluor780 staining). PD-1 and GFP expression in these gated events is shown. Cellular FACS data shows the mean +/− SEM for three independent experiments. (***p

    Journal: Scientific Reports

    Article Title: TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking

    doi: 10.1038/s41598-018-37056-x

    Figure Lengend Snippet: Wild-type Jurkat T cells shed PD-1 upon treatment with ionomycin ( A ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C for 15 minutes then chilled and stained at 4 °C for expression of a variety of surface molecules and analyzed by FACS. Data shows the percentage of initial surface expression after ionomycin treatment. ( B ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining and cellular GFP expression were analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. ( C ) Control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, or a PD-1/GFP chimera where the PD-1 transmembrane sequence was replaced by that from HLA-A2, were treated with 5 μM ionomycin for 15 minutes at 37 °C then chilled and stained at 4 °C for PD-1 expression. PD-1 staining was analyzed by FACS; data shows the percentage of initial expression after ionomycin treatment. (D) Control wild-type Jurkat T cells over-expressing a PD-1/mCherry chimera, were imaged by SR-SIM microscopy before and after ionomycin treatment. Arrows indicate fluorescent vesicles (scale bar is 5 μm). ( E ) Control wild-type Jurkat T cells, or control wild-type Jurkat T cells over-expressing a PD-1/GFP chimera, were stained with an anti-PD-1 antibody, then treated with 5 μM ionomycin for 15 minutes at 37 °C in the presence of fixable viability dye eFluor780. The cell preparations were analyzed by FACS. The events shown were first gated on subcellular sized events (low forward scatter) and intact membranes (low fixable viability dye eFluor780 staining). PD-1 and GFP expression in these gated events is shown. Cellular FACS data shows the mean +/− SEM for three independent experiments. (***p

    Article Snippet: 5 μM ionomycin free acid (Calbiochem) was used unless otherwise stated.

    Techniques: Expressing, Staining, FACS, Sequencing, Microscopy