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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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    93
    Becton Dickinson pma ionomycin
    γδ T cells are a source of IL-17 in brain abscesses Cells were recovered from brain abscesses of C57BL/6 wild type mice at day 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of <t>PMA</t> + <t>ionomycin</t> for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 production in CD4 + Th17 or γδ T cells was quantitated by intracellular cytokine staining. The results presented are representative of two independent experiments.
    Pma Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 847 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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    94
    Becton Dickinson ionomycin
    Th1 cell counts in the presence of ketamine or morphine following PMA and <t>ionomycin</t> stimulation in healthy control and CRC groups. (A) Group 0, healthy control group without PMA and ionomycin treatment. (B) Group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine. (C) Group 2, CRC control group without PMA and ionomycin. (D) Group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine. (E) Group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin. (F) Group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin. (G) Group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin (H) Group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. (I) CD3 + CD8 − IFN-γ + cells, labeled as Th1 cells, in dot plots. Data are presented as the mean ± standard error of the mean. # P
    Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 8343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical ionomycin
    Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after <t>PMA/Ionomycin</t> treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.
    Ionomycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem ionomycin
    Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; <t>PMA/Ionomycin</t> stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P
    Ionomycin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pma ionomycin
    Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; <t>PMA/Ionomycin</t> stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P
    Pma Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1006 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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    99
    Millipore calcium ionophore 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
    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 & 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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    93
    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: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ionomycin ion
    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 Ion, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris ionomycin
    Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with <t>ionomycin.</t> Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p
    Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    92
    Beyotime ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
    Ionomycin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
    Ionomycin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Valiant ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
    Ionomycin, supplied by Valiant, used in various techniques. Bioz Stars score: 94/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology ionomycin
    Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50. ( a ) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with <t>PMA/Ionomycin</t> from Bcl-3 TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. *** P
    Ionomycin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 171 article reviews
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    ionomycin - by Bioz Stars, 2020-10
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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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    97
    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: 97/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    γδ T cells are a source of IL-17 in brain abscesses Cells were recovered from brain abscesses of C57BL/6 wild type mice at day 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 production in CD4 + Th17 or γδ T cells was quantitated by intracellular cytokine staining. The results presented are representative of two independent experiments.

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

    Article Title: Toll-like receptor 2 (TLR2) deficiency leads to increased Th17 infiltrates in experimental brain abscesses †

    doi: 10.4049/jimmunol.0802656

    Figure Lengend Snippet: γδ T cells are a source of IL-17 in brain abscesses Cells were recovered from brain abscesses of C57BL/6 wild type mice at day 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 production in CD4 + Th17 or γδ T cells was quantitated by intracellular cytokine staining. The results presented are representative of two independent experiments.

    Article Snippet: Cells recovered from magnetic cell separation columns were stimulated with a cocktail of PMA + ionomycin (Leukocyte Activation Cocktail) in the presence of GolgiPlug™ for 12 h, whereupon cells were incubated with Fc Block™ (all from BD Biosciences) to minimize non-specific antibody binding.

    Techniques: Mouse Assay, Magnetic Beads, Staining

    The frequency of CD4 + Th17 cells is increased in brain abscesses of TLR2 KO mice T cells were recovered from brain abscesses of TLR2 KO and WT mice at day 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 production was quantitated by intracellular cytokine staining and FACS. The results presented are representative of three independent experiments.

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

    Article Title: Toll-like receptor 2 (TLR2) deficiency leads to increased Th17 infiltrates in experimental brain abscesses †

    doi: 10.4049/jimmunol.0802656

    Figure Lengend Snippet: The frequency of CD4 + Th17 cells is increased in brain abscesses of TLR2 KO mice T cells were recovered from brain abscesses of TLR2 KO and WT mice at day 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 production was quantitated by intracellular cytokine staining and FACS. The results presented are representative of three independent experiments.

    Article Snippet: Cells recovered from magnetic cell separation columns were stimulated with a cocktail of PMA + ionomycin (Leukocyte Activation Cocktail) in the presence of GolgiPlug™ for 12 h, whereupon cells were incubated with Fc Block™ (all from BD Biosciences) to minimize non-specific antibody binding.

    Techniques: Mouse Assay, Magnetic Beads, Staining, FACS

    IL-17-producing cells are more frequent in brain abscesses of TLR2 KO mice T cells were recovered from brain abscesses, draining cervical lymph nodes (CLN), and spleens of TLR2 KO and WT mice at days 7 and 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 and IFN-γ production was quantitated by intracellular cytokine staining and FACS. The results presented are representative of three independent experiments.

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

    Article Title: Toll-like receptor 2 (TLR2) deficiency leads to increased Th17 infiltrates in experimental brain abscesses †

    doi: 10.4049/jimmunol.0802656

    Figure Lengend Snippet: IL-17-producing cells are more frequent in brain abscesses of TLR2 KO mice T cells were recovered from brain abscesses, draining cervical lymph nodes (CLN), and spleens of TLR2 KO and WT mice at days 7 and 14 following S. aureus exposure using CD90-conjugated magnetic beads and stimulated with a cocktail of PMA + ionomycin for 12 h in the presence of GolgiBlock ™ , whereupon IL-17 and IFN-γ production was quantitated by intracellular cytokine staining and FACS. The results presented are representative of three independent experiments.

    Article Snippet: Cells recovered from magnetic cell separation columns were stimulated with a cocktail of PMA + ionomycin (Leukocyte Activation Cocktail) in the presence of GolgiPlug™ for 12 h, whereupon cells were incubated with Fc Block™ (all from BD Biosciences) to minimize non-specific antibody binding.

    Techniques: Mouse Assay, Magnetic Beads, Staining, FACS

    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

    Th1 cell counts in the presence of ketamine or morphine following PMA and ionomycin stimulation in healthy control and CRC groups. (A) Group 0, healthy control group without PMA and ionomycin treatment. (B) Group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine. (C) Group 2, CRC control group without PMA and ionomycin. (D) Group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine. (E) Group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin. (F) Group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin. (G) Group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin (H) Group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. (I) CD3 + CD8 − IFN-γ + cells, labeled as Th1 cells, in dot plots. Data are presented as the mean ± standard error of the mean. # P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Morphine and ketamine treatment suppress the differentiation of T helper cells of patients with colorectal cancer in vitro

    doi: 10.3892/etm.2018.7035

    Figure Lengend Snippet: Th1 cell counts in the presence of ketamine or morphine following PMA and ionomycin stimulation in healthy control and CRC groups. (A) Group 0, healthy control group without PMA and ionomycin treatment. (B) Group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine. (C) Group 2, CRC control group without PMA and ionomycin. (D) Group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine. (E) Group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin. (F) Group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin. (G) Group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin (H) Group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. (I) CD3 + CD8 − IFN-γ + cells, labeled as Th1 cells, in dot plots. Data are presented as the mean ± standard error of the mean. # P

    Article Snippet: Qualifying cells were suspended (1×106 cells) in RPMI Medium1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and incubated for 30 min. PBMCs were then stimulated using 2 µl/ml of a leukocyte activation cocktail containing PMA and ionomycin (P550583; 1X; BD Biosciences, Franklin Lanes, NJ, USA) in the presence or absence of ketamine and morphine, in an atmosphere containing 5% CO2 at 95% humidity and 37°C for 4 h, prior to analysis.

    Techniques: Labeling

    Th1/Th2 ratio in the presence of ketamine or morphine following PMA and ionomycin stimulation in the normal control and CRC groups. Group 0, healthy control group without PMA and ionomycin treatment; group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine; group 2, CRC control group without PMA and ionomycin; group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine; group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin; group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin; group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin; group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. Data are presented as the mean ± standard error of the mean. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Morphine and ketamine treatment suppress the differentiation of T helper cells of patients with colorectal cancer in vitro

    doi: 10.3892/etm.2018.7035

    Figure Lengend Snippet: Th1/Th2 ratio in the presence of ketamine or morphine following PMA and ionomycin stimulation in the normal control and CRC groups. Group 0, healthy control group without PMA and ionomycin treatment; group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine; group 2, CRC control group without PMA and ionomycin; group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine; group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin; group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin; group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin; group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. Data are presented as the mean ± standard error of the mean. *P

    Article Snippet: Qualifying cells were suspended (1×106 cells) in RPMI Medium1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and incubated for 30 min. PBMCs were then stimulated using 2 µl/ml of a leukocyte activation cocktail containing PMA and ionomycin (P550583; 1X; BD Biosciences, Franklin Lanes, NJ, USA) in the presence or absence of ketamine and morphine, in an atmosphere containing 5% CO2 at 95% humidity and 37°C for 4 h, prior to analysis.

    Techniques:

    Th2 cell counts in the presence of ketamine or morphine following PMA and ionomycin stimulation in healthy control and CRC groups. (A) Group 0, healthy control group without PMA and ionomycin treatment. (B) Group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine. (C) Group 2, CRC control group without PMA and ionomycin. (D) Group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine. (E) Group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin. (F) Group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin. (G) Group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin (H) Group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. (I) CD3 + CD8 − IL-4 + cells, labeled as Th2 cells, in dot plots. Data are presented as the mean ± standard error of the mean. # P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Morphine and ketamine treatment suppress the differentiation of T helper cells of patients with colorectal cancer in vitro

    doi: 10.3892/etm.2018.7035

    Figure Lengend Snippet: Th2 cell counts in the presence of ketamine or morphine following PMA and ionomycin stimulation in healthy control and CRC groups. (A) Group 0, healthy control group without PMA and ionomycin treatment. (B) Group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine. (C) Group 2, CRC control group without PMA and ionomycin. (D) Group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine. (E) Group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin. (F) Group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin. (G) Group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin (H) Group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. (I) CD3 + CD8 − IL-4 + cells, labeled as Th2 cells, in dot plots. Data are presented as the mean ± standard error of the mean. # P

    Article Snippet: Qualifying cells were suspended (1×106 cells) in RPMI Medium1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and incubated for 30 min. PBMCs were then stimulated using 2 µl/ml of a leukocyte activation cocktail containing PMA and ionomycin (P550583; 1X; BD Biosciences, Franklin Lanes, NJ, USA) in the presence or absence of ketamine and morphine, in an atmosphere containing 5% CO2 at 95% humidity and 37°C for 4 h, prior to analysis.

    Techniques: Labeling

    Percentages of CD3 + cells in eight groups. Group 0, healthy control group without PMA and ionomycin treatment; group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine; group 2, CRC control group without PMA and ionomycin; group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine; group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin; group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin; group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin; group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. CD3 + , cluster of differentiation 3 + ; PMA, phorbol-myristate-acetate; CRC, colorectal cancer.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Morphine and ketamine treatment suppress the differentiation of T helper cells of patients with colorectal cancer in vitro

    doi: 10.3892/etm.2018.7035

    Figure Lengend Snippet: Percentages of CD3 + cells in eight groups. Group 0, healthy control group without PMA and ionomycin treatment; group 1, healthy patient group treated with PMA and ionomycin but not ketamine or morphine; group 2, CRC control group without PMA and ionomycin; group 3, CRC group treated with PMA and ionomycin but not ketamine or morphine; group 4, CRC group treated with ketamine (25 µM), PMA and ionomycin; group 5, CRC group treated with ketamine (50 µM), PMA and ionomycin; group 6, CRC group treated with ketamine (100 µM), PMA and ionomycin; group 7, CRC group treated with morphine (50 ng/ml), PMA and ionomycin. CD3 + , cluster of differentiation 3 + ; PMA, phorbol-myristate-acetate; CRC, colorectal cancer.

    Article Snippet: Qualifying cells were suspended (1×106 cells) in RPMI Medium1640 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and incubated for 30 min. PBMCs were then stimulated using 2 µl/ml of a leukocyte activation cocktail containing PMA and ionomycin (P550583; 1X; BD Biosciences, Franklin Lanes, NJ, USA) in the presence or absence of ketamine and morphine, in an atmosphere containing 5% CO2 at 95% humidity and 37°C for 4 h, prior to analysis.

    Techniques:

    Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.

    Journal: Frontiers in Microbiology

    Article Title: HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency

    doi: 10.3389/fmicb.2018.01066

    Figure Lengend Snippet: Sense RNA reactivation is influenced by antisense RNA transcription. (A) Representative results of sense (mCherry) and antisense RNA expression (Venus) after PMA/Ionomycin treatment of 2 individual cell clones (AS+2G8 and DN12D7c). Dot plots from flow cytometry at 24 h post stimulation are shown. (B–E) Increase of sense and antisense RNA expression in cloned cells after activation with PMA/Ionomycin (B,C) or SAHA (D,E) . The fold increase of the MFI of mCherry and Venus expression is shown for all individual cell clones. Fold increase was calculated from mCherry or Venus MFI of activated cells using that of non-activated (DMSO treated) cells as reference. Means ± SD of three to five independent experiments are given. P -values were calculated by Mann–Whitney’s tests. (F) Fold sense RNA increase after cell clone activation with PMA/Ionomycin or SAHA. Relative levels of mCherry RNA were measured by sense-strand-specific RT-qPCR. Dots represent PMA/Ionomycin-treated clones; triangles represent SAHA-treated clones. The p value was calculated by Mann–Whitney’s test.

    Article Snippet: For latent rfl-HIV reactivation, PMA (phorbol 12-myristate 13-acetate, Sigma Aldrich) plus Ionomycin (Cayman Chemical, Ann Arbor, MI, United States), or SAHA (Vorinostat, Sigma Aldrich) were used at 20 ng/ml, 1 and 5 μM, respectively.

    Techniques: RNA Expression, Clone Assay, Flow Cytometry, Cytometry, Activation Assay, Expressing, MANN-WHITNEY, Quantitative RT-PCR

    Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; PMA/Ionomycin stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

    Journal: Gastroenterology

    Article Title: Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease

    doi: 10.1053/j.gastro.2018.02.019

    Figure Lengend Snippet: Intrahepatic γδ T Cells Produce IL-17 in Response to Translocated Gut Microbiota. (A-B) Bulk T cells were isolated from Mdr2 −/− livers and spleens via negative selection. Cells were seeded at 2×10 5 cells per well and stimulated for 24 hrs in the presence of media (complete RPMI +20U/mL IL-2), or indicated heat-killed microbial (10 6 /mL) species isolated from Mdr2 −/− livers. T cells were analyzed for IL-17A (A–B; top panels) and IFNγ (A–B; bottom panels) responses. To rule out non-specific TLR pathways, control wells were stimulated with 2μg/mL LPS, or 1μg/mL Pam 3 CSK 4 ; PMA/Ionomycin stimulation served as a positive control (A–B; far right). (C–D) Frequency of IL-17A (C) and IFNγ (D) production by TCRβ- γδTCR+ were compared in the liver versus spleen. Numbers on FACS plots represent the proportion of the TCRβ-γδTCR+ population producing the indicate cytokine. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

    Article Snippet: In some experiments, cells were stimulated with 50ng/mL Phorbol 12-Myristate 13-Acetate (PMA; LC Labs, USA) and 1μg/mL Ionomycin (Enzo Life sciences, USA) for 4 hours in the presence of Golgi Stop/Golgi Plug (BD Biosciences).

    Techniques: Isolation, Selection, Positive Control, FACS, Two Tailed Test, MANN-WHITNEY

    Intrahepatic γδ T Cells Isolated from PSC Patient Livers are Capable of Producing IL-17. (A–B) Bulk liver lymphocytes from human subjects with ESLD as a result of PSC ( n=5 ) or HCV ( n=5) were stimulated overnight with PMA/Ionomycin as described. γδ T cells from PSC patients were capable of IL-17 production (A, left; * P

    Journal: Gastroenterology

    Article Title: Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease

    doi: 10.1053/j.gastro.2018.02.019

    Figure Lengend Snippet: Intrahepatic γδ T Cells Isolated from PSC Patient Livers are Capable of Producing IL-17. (A–B) Bulk liver lymphocytes from human subjects with ESLD as a result of PSC ( n=5 ) or HCV ( n=5) were stimulated overnight with PMA/Ionomycin as described. γδ T cells from PSC patients were capable of IL-17 production (A, left; * P

    Article Snippet: In some experiments, cells were stimulated with 50ng/mL Phorbol 12-Myristate 13-Acetate (PMA; LC Labs, USA) and 1μg/mL Ionomycin (Enzo Life sciences, USA) for 4 hours in the presence of Golgi Stop/Golgi Plug (BD Biosciences).

    Techniques: Isolation

    Fibrotic Liver γδ T Cell Compartment is Altered in Favor of IL-17 Producing Subsets. (A–B) Mdr2 −/− and FVB/N mice were administered 500μg of anti-γδTCR (Clone UC7-13D5) or appropriate Hamster-IgG isotype control intravenously and sacrificed one day following treatment. Livers and spleens were harvested; and bulk lymphocytes were first stained with anti-hamster IgG, followed by TCRVγ1.1–1.2, Vγ2, Vγ3, and for Vγ4, Vγ7 in combination with appropriate surface antibodies. Frequencies were determined based on the percentage of the indicated Vγ-chain within the live non-autofluorescent TCRβ- CD3+ gate (A–B). “Other” refers to in vivo bound and ex vivo detectable UC7-13D5 (A, top; B, Top). (C–D) Lymphocytes were stimulated with PMA/ionomycin in the presence of Golgi Plug/Golgi Stop for 4 hours at 37°C, and subsequently stained for intracellular IL-17A. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

    Journal: Gastroenterology

    Article Title: Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease

    doi: 10.1053/j.gastro.2018.02.019

    Figure Lengend Snippet: Fibrotic Liver γδ T Cell Compartment is Altered in Favor of IL-17 Producing Subsets. (A–B) Mdr2 −/− and FVB/N mice were administered 500μg of anti-γδTCR (Clone UC7-13D5) or appropriate Hamster-IgG isotype control intravenously and sacrificed one day following treatment. Livers and spleens were harvested; and bulk lymphocytes were first stained with anti-hamster IgG, followed by TCRVγ1.1–1.2, Vγ2, Vγ3, and for Vγ4, Vγ7 in combination with appropriate surface antibodies. Frequencies were determined based on the percentage of the indicated Vγ-chain within the live non-autofluorescent TCRβ- CD3+ gate (A–B). “Other” refers to in vivo bound and ex vivo detectable UC7-13D5 (A, top; B, Top). (C–D) Lymphocytes were stimulated with PMA/ionomycin in the presence of Golgi Plug/Golgi Stop for 4 hours at 37°C, and subsequently stained for intracellular IL-17A. Statistical significance was determined by a two-tailed Mann-Whitney Test, * P

    Article Snippet: In some experiments, cells were stimulated with 50ng/mL Phorbol 12-Myristate 13-Acetate (PMA; LC Labs, USA) and 1μg/mL Ionomycin (Enzo Life sciences, USA) for 4 hours in the presence of Golgi Stop/Golgi Plug (BD Biosciences).

    Techniques: Mouse Assay, Staining, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

    Intrahepatic γδ T Cells are Predominant Source of IL-17A During Cholestatic Liver Disease. (A) Liver tissues from 25-wk-old FVB/N and Mdr2 −/− mice were sectioned and stained with Sirius red and H E stain (Mag. 100×). (B) Serum samples were obtained from 25-wk-old Mdr2 −/− and FVB/N mice and the levels of IL-17A were detected using a standard ELISA. (C–D) Bulk lymphocytes isolated from livers and spleens were stimulated with PMA/Ionomycin for 4 hours to determine frequency of live, non-autofluorescent CD3+ (T cells) (C) that produce IL-17A from livers and spleens derived from the indicated group. (D) Quantitation of frequencies of IL-17A+ CD4+ T cells (Top) and γδ T cells (bottom) from livers are shown. Representative figures from more than 3 independent experiments are shown. Two-tailed Mann Whitney test, *** P

    Journal: Gastroenterology

    Article Title: Alterations in Intestinal Microbiota Lead to Production of Interleukin 17 by Intrahepatic γδ T-cell Receptor-positive Cells and Pathogenesis of Cholestatic Liver Disease

    doi: 10.1053/j.gastro.2018.02.019

    Figure Lengend Snippet: Intrahepatic γδ T Cells are Predominant Source of IL-17A During Cholestatic Liver Disease. (A) Liver tissues from 25-wk-old FVB/N and Mdr2 −/− mice were sectioned and stained with Sirius red and H E stain (Mag. 100×). (B) Serum samples were obtained from 25-wk-old Mdr2 −/− and FVB/N mice and the levels of IL-17A were detected using a standard ELISA. (C–D) Bulk lymphocytes isolated from livers and spleens were stimulated with PMA/Ionomycin for 4 hours to determine frequency of live, non-autofluorescent CD3+ (T cells) (C) that produce IL-17A from livers and spleens derived from the indicated group. (D) Quantitation of frequencies of IL-17A+ CD4+ T cells (Top) and γδ T cells (bottom) from livers are shown. Representative figures from more than 3 independent experiments are shown. Two-tailed Mann Whitney test, *** P

    Article Snippet: In some experiments, cells were stimulated with 50ng/mL Phorbol 12-Myristate 13-Acetate (PMA; LC Labs, USA) and 1μg/mL Ionomycin (Enzo Life sciences, USA) for 4 hours in the presence of Golgi Stop/Golgi Plug (BD Biosciences).

    Techniques: Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Isolation, Derivative Assay, Quantitation Assay, Two Tailed Test, MANN-WHITNEY

    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

    Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Exchange Factor EFA6R Requires C-terminal Targeting to the Plasma Membrane to Promote Cytoskeletal Rearrangement through the Activation of ADP-ribosylation Factor 6 (ARF6) *

    doi: 10.1074/jbc.M113.534156

    Figure Lengend Snippet: Requirement of the PH and CC domains for membrane localization of EFA6R. A , upper panel , HeLa cells transfected with GFP-tagged EFA6R constructs were treated with ionomycin. Lower panel , quantification of the ratio of plasma membrane EFA6R to total EFA6R by densitometric analysis. B , the effect of ionomycin and cytochalasin D on subcellular localization is shown schematically. C , upper panel , HeLa cells transfected with GFP-EFA6R were treated with ionomycin, cytochalasin D, or both and fixed, and subcellular localization was visualized by confocal microscopy. Lower panel , quantification of the ratio of plasma membrane-localized EFA6R to total EFA6R by densitometric analysis. ***, p

    Article Snippet: Ionomycin and Rapamycin Treatments 2 days after transfection, cells were washed three times with PBS and stimulated with either 10 μm ionomycin (Tocris) for 5 min in Hanks' balanced salt solution or 100 nm rapamycin (LC Laboratories) for 3 min in PBS at 37 °C.

    Techniques: Transfection, Construct, Confocal Microscopy

    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

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

    Journal: Chinese Medical Journal

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

    doi: 10.4103/0366-6999.223855

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

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

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

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

    Journal: Chinese Medical Journal

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

    doi: 10.4103/0366-6999.223855

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

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

    Techniques: Expressing, Transfection, Activity Assay

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

    Journal: Chinese Medical Journal

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

    doi: 10.4103/0366-6999.223855

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

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

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

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

    Journal: Chinese Medical Journal

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

    doi: 10.4103/0366-6999.223855

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

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

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

    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:

    Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50. ( a ) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with PMA/Ionomycin from Bcl-3 TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. *** P

    Journal: Nature Communications

    Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

    doi: 10.1038/ncomms15069

    Figure Lengend Snippet: Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50. ( a ) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with PMA/Ionomycin from Bcl-3 TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. *** P

    Article Snippet: Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml−1 , Santa Cruz) and Ionomycin (1 μg ml−1 , Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910).

    Techniques: Activity Assay, Binding Assay, Construct, In Vitro, Mouse Assay, Luciferase, Plasmid Preparation, Transfection

    Bcl-3 TOE Tregs display diminished suppressive capacity. ( a ) Flow cytometric analysis of MACS-purified CD4 + CD25 + Treg cells of the indicated genotypes restimulated with PMA/Ionomycin and BrefeldinA for 4 h. Cells are gated on Foxp3 + and analysed for IL-10 expression. n =3. ( b ) Quantitative RT–PCR of MACS purified CD4 + CD25 + Treg cells from the indicated mice ( n =3). Shown are transcription counts of the indicated genes normalized to HPRT and littermate controls. * P

    Journal: Nature Communications

    Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

    doi: 10.1038/ncomms15069

    Figure Lengend Snippet: Bcl-3 TOE Tregs display diminished suppressive capacity. ( a ) Flow cytometric analysis of MACS-purified CD4 + CD25 + Treg cells of the indicated genotypes restimulated with PMA/Ionomycin and BrefeldinA for 4 h. Cells are gated on Foxp3 + and analysed for IL-10 expression. n =3. ( b ) Quantitative RT–PCR of MACS purified CD4 + CD25 + Treg cells from the indicated mice ( n =3). Shown are transcription counts of the indicated genes normalized to HPRT and littermate controls. * P

    Article Snippet: Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml−1 , Santa Cruz) and Ionomycin (1 μg ml−1 , Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910).

    Techniques: Flow Cytometry, Magnetic Cell Separation, Purification, Expressing, Quantitative RT-PCR, Mouse Assay

    Bcl-3 prevents p50 binding to the Foxp3 and CTLA-4 promoter. ( a ) Western blot analysis of Bcl-3 and the NF-κB family members p50, p52 and p65 in nuclear versus whole extract of Bcl-3 TOE and littermate controls using Tregs either unstimulated (left) or restimulated for 2 h with PMA/Ionomycin (right). One representative blot from two independent experiments is shown. ( b ) Immunoprecipitation with Bcl-3 antibodies using protein extracts from in vitro differentiated Tregs, restimulated with PMA/Ionomycin for 4 h and followed by immunoblot analysis with antibodies against Bcl-3 or p50. One representative blot from two independent experiments is shown. ( c ) Pull-down experiments with DNA probes from promoters of IL2Ra, Foxp3 CN2 (from top to bottowm) with protein extracts from Tregs to which Bcl-3 or empty vector overexpressing lysates were added. One out of two experiments is demonstrated. ( d ) CHiP assay using p50 and isotope control antibodies antibodies were performed in in vitro differentiated Tregs from Bcl3 TOE mice and littermate controls. Primer were designed as indicated for two sites within the CTLA-4 and Foxp3 Promoter region. One of two representative biological examples is shown.

    Journal: Nature Communications

    Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

    doi: 10.1038/ncomms15069

    Figure Lengend Snippet: Bcl-3 prevents p50 binding to the Foxp3 and CTLA-4 promoter. ( a ) Western blot analysis of Bcl-3 and the NF-κB family members p50, p52 and p65 in nuclear versus whole extract of Bcl-3 TOE and littermate controls using Tregs either unstimulated (left) or restimulated for 2 h with PMA/Ionomycin (right). One representative blot from two independent experiments is shown. ( b ) Immunoprecipitation with Bcl-3 antibodies using protein extracts from in vitro differentiated Tregs, restimulated with PMA/Ionomycin for 4 h and followed by immunoblot analysis with antibodies against Bcl-3 or p50. One representative blot from two independent experiments is shown. ( c ) Pull-down experiments with DNA probes from promoters of IL2Ra, Foxp3 CN2 (from top to bottowm) with protein extracts from Tregs to which Bcl-3 or empty vector overexpressing lysates were added. One out of two experiments is demonstrated. ( d ) CHiP assay using p50 and isotope control antibodies antibodies were performed in in vitro differentiated Tregs from Bcl3 TOE mice and littermate controls. Primer were designed as indicated for two sites within the CTLA-4 and Foxp3 Promoter region. One of two representative biological examples is shown.

    Article Snippet: Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml−1 , Santa Cruz) and Ionomycin (1 μg ml−1 , Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910).

    Techniques: Binding Assay, Western Blot, Immunoprecipitation, In Vitro, Plasmid Preparation, Chromatin Immunoprecipitation, Mouse Assay

    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

    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