ionomycin  (Abcam)

 
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    Name:
    Ionomycin Ca
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    Catalog Number:
    ab120116
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    Structured Review

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

    https://www.bioz.com/result/ionomycin/product/Abcam
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    ionomycin - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells"

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

    Journal: Nitric oxide : biology and chemistry

    doi: 10.1016/j.niox.2017.09.001

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

    Techniques Used: Derivative Assay

    2) Product Images from "Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+"

    Article Title: Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+

    Journal: Scientific Reports

    doi: 10.1038/srep15602

    Ca 2+ dynamically controls rearrangement of MICU1 multimers in intact cells. ( A ) Schematic illustration of the putative Ca 2+ -induced rearrangement of MICU1-CFP and MICU1-YFP hexamers that bind to the MCU/EMRE mitochondrial Ca 2+ uptake machinery. It is hypothesized that Ca 2+ binding to the MICU1 EF-hands reduces FRET between the respective MICU1-conjugated FPs. ( B ) Representative curve showing the MICU1 FRET ratio signal of HeLa cells co-expressing MICU1-CFP and MICU1-YFP upon cell treatment with 100 μM histamine in the presence of 2 mM Ca 2+ . ( C ) Representative images to panel b under basal condition, upon stimulation with 100 μM histamine 3 min. after stimulation (scale bar, 10 μm). ( D ) Representative MICU1 FRET ratio signals over time in HeLa cells upon addition and removal of different free Ca 2+ concentrations in the presence of 3 μM ionomycin. The maximal ∆ MICU1 FRET ratio signal (between 3 mM EGTA and 1000 μM Ca 2+ ) was defined as 100%. ( E ) Concentration response curve of the Ca 2+ -induced reduction of the MICU1 FRET ratio signal in HeLa cells that were treated with 3 μM ionomycin and different Ca 2+ concentrations as shown in panel ( D ); mean ± SEM. (n = 7–9).
    Figure Legend Snippet: Ca 2+ dynamically controls rearrangement of MICU1 multimers in intact cells. ( A ) Schematic illustration of the putative Ca 2+ -induced rearrangement of MICU1-CFP and MICU1-YFP hexamers that bind to the MCU/EMRE mitochondrial Ca 2+ uptake machinery. It is hypothesized that Ca 2+ binding to the MICU1 EF-hands reduces FRET between the respective MICU1-conjugated FPs. ( B ) Representative curve showing the MICU1 FRET ratio signal of HeLa cells co-expressing MICU1-CFP and MICU1-YFP upon cell treatment with 100 μM histamine in the presence of 2 mM Ca 2+ . ( C ) Representative images to panel b under basal condition, upon stimulation with 100 μM histamine 3 min. after stimulation (scale bar, 10 μm). ( D ) Representative MICU1 FRET ratio signals over time in HeLa cells upon addition and removal of different free Ca 2+ concentrations in the presence of 3 μM ionomycin. The maximal ∆ MICU1 FRET ratio signal (between 3 mM EGTA and 1000 μM Ca 2+ ) was defined as 100%. ( E ) Concentration response curve of the Ca 2+ -induced reduction of the MICU1 FRET ratio signal in HeLa cells that were treated with 3 μM ionomycin and different Ca 2+ concentrations as shown in panel ( D ); mean ± SEM. (n = 7–9).

    Techniques Used: Binding Assay, Expressing, Concentration Assay

    The Ca 2+ -induced rearrangement of MICU1 multimers is independent of the expression level of MCU and EMRE. ( A ) Average curves of MICU1 FRET ratio signals over time in response to 100 μM histamine in Ca 2+ -free solution of control HeLa cells (black curve, n = 23) and cells treated with siRNA against MCU (blue curve, n = 22). ( B ) Bars represent maximal ∆ MICU1 FRET ratio values (mean ± SEM) extracted from curves shown in panel A. ( C ) Bars represent maximal ∆ MICU1 FRET ratio values upon cell treatment with 100 μM histamine in Ca 2+ -free solution of control HeLa cells (white column, n = 19) and cells treated with siRNA against EMRE (red column, n = 12). ( D ) Concentration response curves showing the effects of different Ca 2+ concentrations on the MICU1 FRET ratio in ionomycin- (3 μM) treated control HeLa cells (black curve, white circles, n = 7–9), cells reduced of MCU (blue dotted curve, blue filled squares, n = 8–10), and cells reduced of EMRE (red dotted curve, r ed filled rhombs, n = 10).
    Figure Legend Snippet: The Ca 2+ -induced rearrangement of MICU1 multimers is independent of the expression level of MCU and EMRE. ( A ) Average curves of MICU1 FRET ratio signals over time in response to 100 μM histamine in Ca 2+ -free solution of control HeLa cells (black curve, n = 23) and cells treated with siRNA against MCU (blue curve, n = 22). ( B ) Bars represent maximal ∆ MICU1 FRET ratio values (mean ± SEM) extracted from curves shown in panel A. ( C ) Bars represent maximal ∆ MICU1 FRET ratio values upon cell treatment with 100 μM histamine in Ca 2+ -free solution of control HeLa cells (white column, n = 19) and cells treated with siRNA against EMRE (red column, n = 12). ( D ) Concentration response curves showing the effects of different Ca 2+ concentrations on the MICU1 FRET ratio in ionomycin- (3 μM) treated control HeLa cells (black curve, white circles, n = 7–9), cells reduced of MCU (blue dotted curve, blue filled squares, n = 8–10), and cells reduced of EMRE (red dotted curve, r ed filled rhombs, n = 10).

    Techniques Used: Expressing, Concentration Assay

    3) Product Images from "Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates"

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

    Journal: Haematologica

    doi: 10.3324/haematol.2018.195057

    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
    Figure Legend 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

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

    4) Product Images from "Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling"

    Article Title: Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.01544

    Ca 2+ dynamics induced by (A,B) ionomycin, (C,D) CPA, (E,F) ionomycin after CPA-induced ER depletion in neurons dissociated from larval brains expressing spastin K467R , and relative controls, detected by fura-2. (A,C,E) Cells were stimulated with (A,E) 10 μM ionomycin or (C) 50 μM CPA in the presence of extracellular Ca 2+ chelator EGTA. In all panels, average traces are represented as R/R 0 values of n > 100 cells. (B,D,F) Histograms report the average area under curve (AUC) values of the traces recorded. Data are presented as mean ± SEM of n > 100 cells. *** p
    Figure Legend Snippet: Ca 2+ dynamics induced by (A,B) ionomycin, (C,D) CPA, (E,F) ionomycin after CPA-induced ER depletion in neurons dissociated from larval brains expressing spastin K467R , and relative controls, detected by fura-2. (A,C,E) Cells were stimulated with (A,E) 10 μM ionomycin or (C) 50 μM CPA in the presence of extracellular Ca 2+ chelator EGTA. In all panels, average traces are represented as R/R 0 values of n > 100 cells. (B,D,F) Histograms report the average area under curve (AUC) values of the traces recorded. Data are presented as mean ± SEM of n > 100 cells. *** p

    Techniques Used: Expressing

    5) Product Images from "Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells"

    Article Title: Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells

    Journal: Nature Communications

    doi: 10.1038/ncomms11226

    Ndfip2 deficiency exacerbates inflammation in Ndfip1 fl/fl CD4Cre+ mice. ( a ) H E-stained sections of oesophagus, lung and skin from representative 8-week-old control, Ndfip1 fl/fl CD4 Cre+ (cKO) and Ndfip2 −/− Ndfip1 fl/fl CD4 Cre+ (cDKO) mice (bar represents 100 μm). ( b , c ) Body weight ( b ) and ( c ) spleen count of 5–7-week-old WT, cKO, Ndfip2 −/− and cDKO mice. Mean±s.e.m. n =5–15 mice. ( d , e ) Representative flow cytometry analysis of splenic CD3+CD4+ T cells, showing ( d ) expression CD44 and CD62L and ( e ) intracellular levels of IL-4 after ex vivo stimulation with PMA/ionomycin in the presence of BFA. ( f – i ) Quantification of ( f ) the number of CD4+ T cells, ( g ) naive CD62L high CD4+ T cells, ( h ) CD44+ CD4+ T cells and ( i ) IL-4+ CD4s from spleen analysed by flow cytometry. Mean±s.e.m., n =7–12 mice. ( j , k ) Quantification of percent IL-4+ or IFNγ+ CD4+ T cells among CD44 high T cells. Mean±s.e.m., n =4–7 mice. P values calculated by one-way ANOVA with Holm–Sidak test for multiple comparisons: * P
    Figure Legend Snippet: Ndfip2 deficiency exacerbates inflammation in Ndfip1 fl/fl CD4Cre+ mice. ( a ) H E-stained sections of oesophagus, lung and skin from representative 8-week-old control, Ndfip1 fl/fl CD4 Cre+ (cKO) and Ndfip2 −/− Ndfip1 fl/fl CD4 Cre+ (cDKO) mice (bar represents 100 μm). ( b , c ) Body weight ( b ) and ( c ) spleen count of 5–7-week-old WT, cKO, Ndfip2 −/− and cDKO mice. Mean±s.e.m. n =5–15 mice. ( d , e ) Representative flow cytometry analysis of splenic CD3+CD4+ T cells, showing ( d ) expression CD44 and CD62L and ( e ) intracellular levels of IL-4 after ex vivo stimulation with PMA/ionomycin in the presence of BFA. ( f – i ) Quantification of ( f ) the number of CD4+ T cells, ( g ) naive CD62L high CD4+ T cells, ( h ) CD44+ CD4+ T cells and ( i ) IL-4+ CD4s from spleen analysed by flow cytometry. Mean±s.e.m., n =7–12 mice. ( j , k ) Quantification of percent IL-4+ or IFNγ+ CD4+ T cells among CD44 high T cells. Mean±s.e.m., n =4–7 mice. P values calculated by one-way ANOVA with Holm–Sidak test for multiple comparisons: * P

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing, Ex Vivo

    Ndfip1/Ndfip2-deficient CD4+ T cells cause increased colitis. ( a – e ) 0.5 × 10 6 sorted naive CD4+ T cells from WT, Ndfip2 −/−, cKO and cDKO mice were transferred into 6-week-old Rag1 −/− recipients. Mice were weighed twice weekly and killed 6 weeks after transfer when 20% weight loss was observed in multiple mice. Spleen weight and body weight were compared to generate an inflammation index ( a ) and colons were measured ( b ). H E-stained sections of the distal colon were imaged on the × 20 objective, and crypt depth was quantified ( c , d ). Splenocytes were stained for intracellular IL-4 and IL-17 after ex vivo stimulation with PMA/ionomycin in the presence of BFA, and analysed by flow cytometry ( e ). Previously gated on live singlets, CD4+, dump gate-. Quantifications shown ±s.e.m. n =5–7 mice. Control (ctrl) mice did not receive T cells. P values calculated by ordinary one-way ANOVA with Holm–Sidak test for multiple comparisons: * P
    Figure Legend Snippet: Ndfip1/Ndfip2-deficient CD4+ T cells cause increased colitis. ( a – e ) 0.5 × 10 6 sorted naive CD4+ T cells from WT, Ndfip2 −/−, cKO and cDKO mice were transferred into 6-week-old Rag1 −/− recipients. Mice were weighed twice weekly and killed 6 weeks after transfer when 20% weight loss was observed in multiple mice. Spleen weight and body weight were compared to generate an inflammation index ( a ) and colons were measured ( b ). H E-stained sections of the distal colon were imaged on the × 20 objective, and crypt depth was quantified ( c , d ). Splenocytes were stained for intracellular IL-4 and IL-17 after ex vivo stimulation with PMA/ionomycin in the presence of BFA, and analysed by flow cytometry ( e ). Previously gated on live singlets, CD4+, dump gate-. Quantifications shown ±s.e.m. n =5–7 mice. Control (ctrl) mice did not receive T cells. P values calculated by ordinary one-way ANOVA with Holm–Sidak test for multiple comparisons: * P

    Techniques Used: Mouse Assay, Staining, Ex Vivo, Flow Cytometry, Cytometry

    Ndfip2 −/− mice do not show signs of inflammation. ( a , b ) Representative flow cytometry analysis of T-cell populations from thymus and spleen of 5–7-week-old Ndfip2 −/− and age-matched control mice: ( a ) CD4+ and CD8+ cells, ( b ) CD44 and CD62L expression on these cells, as noted. ( c ) Intracellular cytokine staining for IL-4 and IFNγ in CD4+ T cells from Ndfip2 −/− and WT spleens stimulated ex vivo with PMA and ionomycin in the presence of BFA. Representative of at least five mice per genotype, 5–7 weeks of age. ( d ) CD4+ T cells were stimulated in vitro for the indicated time periods with αCD3/CD28. Ndfip1 and Ndfip2 expression was analysed by qPCR. Ndfip1/Ndfip2 expression relative to Actb was normalized to expression in unstimulated CD4+ T cells. Representative of a minimum of three independent experiments.
    Figure Legend Snippet: Ndfip2 −/− mice do not show signs of inflammation. ( a , b ) Representative flow cytometry analysis of T-cell populations from thymus and spleen of 5–7-week-old Ndfip2 −/− and age-matched control mice: ( a ) CD4+ and CD8+ cells, ( b ) CD44 and CD62L expression on these cells, as noted. ( c ) Intracellular cytokine staining for IL-4 and IFNγ in CD4+ T cells from Ndfip2 −/− and WT spleens stimulated ex vivo with PMA and ionomycin in the presence of BFA. Representative of at least five mice per genotype, 5–7 weeks of age. ( d ) CD4+ T cells were stimulated in vitro for the indicated time periods with αCD3/CD28. Ndfip1 and Ndfip2 expression was analysed by qPCR. Ndfip1/Ndfip2 expression relative to Actb was normalized to expression in unstimulated CD4+ T cells. Representative of a minimum of three independent experiments.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, Ex Vivo, In Vitro, Real-time Polymerase Chain Reaction

    6) Product Images from "Calcium signalling links MYC to NUAK1"

    Article Title: Calcium signalling links MYC to NUAK1

    Journal: Oncogene

    doi: 10.1038/onc.2017.394

    Calcium signalling activates NUAK1. ( a ) Lysates from HeLa cells treated with the indicated concentrations of HTH-01-015 for 1 h and probed for phospho- and total MYPT1. ( b ) Lysates from HeLa cells transfected with NUAK1 siRNA and probed with the indicated antibodies. nt, non-targeting control siRNA. ( c ) Lysates from HeLa cells transfected with NUAK2 (+) or control (−) siRNA and treated ±10 μ m HTH-01-015, as indicated. Densitometry shows phospho-MYPT1 levels from the image shown. ( d ) Lysates from HeLa cells pre-treated with 5 μg/ml STO-609 for 1 h prior to stimulation with 3 μ m A23187 (10 min) as indicated, and probed with the indicated antibodies. ( e ) Lysates from HeLa cells pre-treated with 10 μ m HTH-01-015 for 1 h prior to stimulation with 3 μ m A23187 or Ionomycin (both 10 min) as indicated, and probed for phosphor-MYPT1. ( f ) Lysates from HeLa cells treated ±20 μ m BAPTA for 30 min. All images are representative of at least three independent experiments, except ( f ) where N =2.
    Figure Legend Snippet: Calcium signalling activates NUAK1. ( a ) Lysates from HeLa cells treated with the indicated concentrations of HTH-01-015 for 1 h and probed for phospho- and total MYPT1. ( b ) Lysates from HeLa cells transfected with NUAK1 siRNA and probed with the indicated antibodies. nt, non-targeting control siRNA. ( c ) Lysates from HeLa cells transfected with NUAK2 (+) or control (−) siRNA and treated ±10 μ m HTH-01-015, as indicated. Densitometry shows phospho-MYPT1 levels from the image shown. ( d ) Lysates from HeLa cells pre-treated with 5 μg/ml STO-609 for 1 h prior to stimulation with 3 μ m A23187 (10 min) as indicated, and probed with the indicated antibodies. ( e ) Lysates from HeLa cells pre-treated with 10 μ m HTH-01-015 for 1 h prior to stimulation with 3 μ m A23187 or Ionomycin (both 10 min) as indicated, and probed for phosphor-MYPT1. ( f ) Lysates from HeLa cells treated ±20 μ m BAPTA for 30 min. All images are representative of at least three independent experiments, except ( f ) where N =2.

    Techniques Used: Transfection

    NUAK1 regulates RAPTOR via AMPK-dependent and independent mechanisms. ( a ) Measurement of protein synthesis (methionine incorporation) in HeLa (left panel) and U2OS (right panel) cells transfected with non-targeting (−), NUAK1 and PKCα siRNA. Mean and s.d. from three independent experiments shown. Statistical significance was determined by one-tailed unpaired T -test. ( b ) Lysates from U2OS cells pre-treated with 10 μ m HTH-01-015 for 1 h, where indicated, prior to treatment with 6 μ m A23187 (10 min), 10 m m salicylate (1 h), 10 m m phenformin (1 h) or DMSO vehicle and blotted for phospho-S792 and total RAPTOR. N =3. The asterisk denotes a nonspecific band in the p-RAPTOR panel ( b , c ). ( c ) Lysates from U2OS cells transfected where indicated with siRNA targeting NUAK1 and treated with 10 m m salicylate (1 h), 10 m m phenformin (1 h), 6 μ m A23187 (10 min), 3 μ m Ionomycin (10 min) or DMSO vehicle, blotted for p-RAPTOR S792 . N =3. ( d ) Lysates from immortalized Prkaa1 FL/FL ;Prkaa2 FL/FL double floxed MEFs, infected overnight with Adeno-LacZ or Adeno-CRE and treated as per ( c ) with AMPK activators in the presence or absence of 10 μ m HTH-01-015, blotted with the indicated antibodies. N =2. ( e ) Lysates from primary Nuak1 FL/FL MEFs stably expressing Cre-ER were treated overnight with 100 n m 4-OH-Tamoxifen (+) or vehicle control (−) prior to stimulation as per ( d , e ) with AMPK activators, then immunoblotted for p-Raptor S792 . N =2.
    Figure Legend Snippet: NUAK1 regulates RAPTOR via AMPK-dependent and independent mechanisms. ( a ) Measurement of protein synthesis (methionine incorporation) in HeLa (left panel) and U2OS (right panel) cells transfected with non-targeting (−), NUAK1 and PKCα siRNA. Mean and s.d. from three independent experiments shown. Statistical significance was determined by one-tailed unpaired T -test. ( b ) Lysates from U2OS cells pre-treated with 10 μ m HTH-01-015 for 1 h, where indicated, prior to treatment with 6 μ m A23187 (10 min), 10 m m salicylate (1 h), 10 m m phenformin (1 h) or DMSO vehicle and blotted for phospho-S792 and total RAPTOR. N =3. The asterisk denotes a nonspecific band in the p-RAPTOR panel ( b , c ). ( c ) Lysates from U2OS cells transfected where indicated with siRNA targeting NUAK1 and treated with 10 m m salicylate (1 h), 10 m m phenformin (1 h), 6 μ m A23187 (10 min), 3 μ m Ionomycin (10 min) or DMSO vehicle, blotted for p-RAPTOR S792 . N =3. ( d ) Lysates from immortalized Prkaa1 FL/FL ;Prkaa2 FL/FL double floxed MEFs, infected overnight with Adeno-LacZ or Adeno-CRE and treated as per ( c ) with AMPK activators in the presence or absence of 10 μ m HTH-01-015, blotted with the indicated antibodies. N =2. ( e ) Lysates from primary Nuak1 FL/FL MEFs stably expressing Cre-ER were treated overnight with 100 n m 4-OH-Tamoxifen (+) or vehicle control (−) prior to stimulation as per ( d , e ) with AMPK activators, then immunoblotted for p-Raptor S792 . N =2.

    Techniques Used: Transfection, One-tailed Test, Infection, Stable Transfection, Expressing

    7) Product Images from "Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells"

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04418-w

    Dynamin-2-CNM-causing mutations reduce stimulus-induced translocation of endogenous GLUT4 in RCMH myoblasts. RCMH myoblasts were transfected with the dynamin-2 EGFP-fused constructs WT, K44A, R369W, R465W or R522H. 48 h later, transfected cells were stimulated with 20 μM ionomycin during 5 min, fixed, immunolabeled with a monoclonal antibody directed against GLUT4 and visualized by TIRF microscopy. Plasma membrane insertion of endogenous GLUT4 was quantified as the total intensity fluorescence of GLUT4 in the evanescent field. ( a , b ) Examples of TIRF images (250 × 250 pixels) of RCMH myoblasts at the resting ( a ) and ionomycin-stimulated condition ( b ). Cell periphery is drawn in white in GLUT4 panels. ( c ) Quantification of GLUT4 total intensity fluorescence in the evanescent field. Note that cells expressing K44A mutant exhibit significantly higher levels of GLUT4 at the resting condition compared to resting-cells expressing Dyn2WT. Ionomycin increased GLUT4 signal in Dyn2WT-transfected cells but was not enough to increase GLUT4 signal in myoblasts expressing all the mutated versions of dynamin-2. Data are expressed as the mean GLUT4 total intensity fluorescence ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance compared to Dyn2WT-transfected resting cells and Dyn2WT-transfected stimulated cells respectively. N is between 18 and 35 cells, from at least three different cultures per experimental condition.
    Figure Legend Snippet: Dynamin-2-CNM-causing mutations reduce stimulus-induced translocation of endogenous GLUT4 in RCMH myoblasts. RCMH myoblasts were transfected with the dynamin-2 EGFP-fused constructs WT, K44A, R369W, R465W or R522H. 48 h later, transfected cells were stimulated with 20 μM ionomycin during 5 min, fixed, immunolabeled with a monoclonal antibody directed against GLUT4 and visualized by TIRF microscopy. Plasma membrane insertion of endogenous GLUT4 was quantified as the total intensity fluorescence of GLUT4 in the evanescent field. ( a , b ) Examples of TIRF images (250 × 250 pixels) of RCMH myoblasts at the resting ( a ) and ionomycin-stimulated condition ( b ). Cell periphery is drawn in white in GLUT4 panels. ( c ) Quantification of GLUT4 total intensity fluorescence in the evanescent field. Note that cells expressing K44A mutant exhibit significantly higher levels of GLUT4 at the resting condition compared to resting-cells expressing Dyn2WT. Ionomycin increased GLUT4 signal in Dyn2WT-transfected cells but was not enough to increase GLUT4 signal in myoblasts expressing all the mutated versions of dynamin-2. Data are expressed as the mean GLUT4 total intensity fluorescence ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance compared to Dyn2WT-transfected resting cells and Dyn2WT-transfected stimulated cells respectively. N is between 18 and 35 cells, from at least three different cultures per experimental condition.

    Techniques Used: Translocation Assay, Transfection, Construct, Immunolabeling, Microscopy, Fluorescence, Expressing, Mutagenesis

    Pharmacological inhibition of dynamin GTP-activity reduces GLUT4 insertion in the plasma membrane of RCMH myoblasts. Cultured RCMH myoblasts were transfected with the GFP-GLUT4-HA construct and 48 ± 2 h later were stimulated for 5 min with 20 µM ionomycin in the presence of 20 µM dynole 31-2, 20 µM dynole 34-2, 4 µM CytoD or the vehicle DMSO at 37 °C, fixed, immunolabeled with an anti-HA antibody without permeabilization, incubated with a Cy3-conjugated antibody and visualized by TIRF microscopy. The plasma membrane insertion of GLUT4 was quantified as a ratio between Cy3 (red) and GFP (green) signals in the evanescent field. ( a ) Schematic representation of the GFP-GLUT4-HA construct; GFP is located in the N-terminal intracellular loop and the exofacial hemaglutinin (HA)-tag in the first extracellular loop. ( b ) Examples of TIRF images of non-treated cells under resting (upper panels) or stimulated (bottom panels) conditions (250 × 250 pixels images are shown). Note that ionomycin induces GLUT4 translocation, as the HA (Cy3) signal in the evanescent field becomes detectable (red spots). ( d ) 250 × 250 pixels representative images of RCMH cells stimulated in the presence of DMSO, CytoD, dynole 31-2 or dynole 34-2. Cell periphery is drawn in white. ( e ) HA (Cy3)/GFP ratio in the evanescent field is plotted for each experimental condition. Note that, as CytoD does, dynole 34-2 significantly inhibits the stimulus-dependent plasma membrane insertion of GLUT4. Data are means ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance compared to the respective resting condition; and # symbols denote significance compared to ionomycin-stimulated cells treated with DMSO and Dynole 31-2, respectively. N is between 20 and 29 cells, from at least three different cultures per experimental condition. Scale bar = 10 µm.
    Figure Legend Snippet: Pharmacological inhibition of dynamin GTP-activity reduces GLUT4 insertion in the plasma membrane of RCMH myoblasts. Cultured RCMH myoblasts were transfected with the GFP-GLUT4-HA construct and 48 ± 2 h later were stimulated for 5 min with 20 µM ionomycin in the presence of 20 µM dynole 31-2, 20 µM dynole 34-2, 4 µM CytoD or the vehicle DMSO at 37 °C, fixed, immunolabeled with an anti-HA antibody without permeabilization, incubated with a Cy3-conjugated antibody and visualized by TIRF microscopy. The plasma membrane insertion of GLUT4 was quantified as a ratio between Cy3 (red) and GFP (green) signals in the evanescent field. ( a ) Schematic representation of the GFP-GLUT4-HA construct; GFP is located in the N-terminal intracellular loop and the exofacial hemaglutinin (HA)-tag in the first extracellular loop. ( b ) Examples of TIRF images of non-treated cells under resting (upper panels) or stimulated (bottom panels) conditions (250 × 250 pixels images are shown). Note that ionomycin induces GLUT4 translocation, as the HA (Cy3) signal in the evanescent field becomes detectable (red spots). ( d ) 250 × 250 pixels representative images of RCMH cells stimulated in the presence of DMSO, CytoD, dynole 31-2 or dynole 34-2. Cell periphery is drawn in white. ( e ) HA (Cy3)/GFP ratio in the evanescent field is plotted for each experimental condition. Note that, as CytoD does, dynole 34-2 significantly inhibits the stimulus-dependent plasma membrane insertion of GLUT4. Data are means ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance compared to the respective resting condition; and # symbols denote significance compared to ionomycin-stimulated cells treated with DMSO and Dynole 31-2, respectively. N is between 20 and 29 cells, from at least three different cultures per experimental condition. Scale bar = 10 µm.

    Techniques Used: Inhibition, Activity Assay, Cell Culture, Transfection, Construct, Immunolabeling, Incubation, Microscopy, Translocation Assay

    8) Product Images from "Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia"

    Article Title: Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2016.69

    Itch −/− CD4 + T cells are sufficient to drive loss of alveolar macrophages in an IL-4-dependent manner. ( a ) Diagram describing experimental design. A total of 4 × 10 6 purified spleen CD4 + T cells from WT, Itch −/− or Itch −/− IL4 −/− mice were transferred intravenously to Rag −/− mice, then BAL and lungs were collected 8 weeks later. ( b ) Lung cell suspensions were stimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A, and then cells were stained for surface markers and intracellular cytokines. Representative flow cytometry plots and quantifications of absolute numbers are shown. Cells are gated on live, singlet, CD3 + CD4 + . Dots represent individual mice ( n =5–6). ( c ) Alveolar macrophages were identified from lung cell suspensions by flow cytometry. Representative flow cytometry plots and quantification of alveolar macrophages are shown. Flow plots were gated on live, singlet, CD45 + , and alveolar macrophages were CD11c + CD11b lo SiglecF + . ( d ) The mean fluorescence intensity of the forward- and side-scatter parameters (FSC and SSC, respectively) was calculated for alveolar macrophages using the geometric mean formula on FlowJo software. Alveolar macrophage numbers, FSC and SSC were divided by the average WT numbers within each experiment to calculate fold change per experiment, normalizing for experimental variability ( n =5–6, compiled from two independent experiments). ( e ) Representative BAL cytospins stained with modified Giemsa stain and visualized at × 40. Significance was calculated using a one-way analysis of variance. * or ** denote P ≤0.05 or P ≤0.01, respectively.
    Figure Legend Snippet: Itch −/− CD4 + T cells are sufficient to drive loss of alveolar macrophages in an IL-4-dependent manner. ( a ) Diagram describing experimental design. A total of 4 × 10 6 purified spleen CD4 + T cells from WT, Itch −/− or Itch −/− IL4 −/− mice were transferred intravenously to Rag −/− mice, then BAL and lungs were collected 8 weeks later. ( b ) Lung cell suspensions were stimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A, and then cells were stained for surface markers and intracellular cytokines. Representative flow cytometry plots and quantifications of absolute numbers are shown. Cells are gated on live, singlet, CD3 + CD4 + . Dots represent individual mice ( n =5–6). ( c ) Alveolar macrophages were identified from lung cell suspensions by flow cytometry. Representative flow cytometry plots and quantification of alveolar macrophages are shown. Flow plots were gated on live, singlet, CD45 + , and alveolar macrophages were CD11c + CD11b lo SiglecF + . ( d ) The mean fluorescence intensity of the forward- and side-scatter parameters (FSC and SSC, respectively) was calculated for alveolar macrophages using the geometric mean formula on FlowJo software. Alveolar macrophage numbers, FSC and SSC were divided by the average WT numbers within each experiment to calculate fold change per experiment, normalizing for experimental variability ( n =5–6, compiled from two independent experiments). ( e ) Representative BAL cytospins stained with modified Giemsa stain and visualized at × 40. Significance was calculated using a one-way analysis of variance. * or ** denote P ≤0.05 or P ≤0.01, respectively.

    Techniques Used: Purification, Mouse Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, Software, Modification, Giemsa Stain

    9) Product Images from "Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection"

    Article Title: Immune Activation Influences SAMHD1 Expression and Vpx-mediated SAMHD1 Degradation during Chronic HIV-1 Infection

    Journal: Scientific Reports

    doi: 10.1038/srep38162

    The effects of immune activation on SAMHD1 expression and SIV-Vpx mediated SAMHD1 degradation. ( a ) PBMC cells were stimulated with PI (1 μM PMA plus 1 μg/mL Ionomycin) for 24 h. Then, SAMHD1 expression in CD4 + T cells was assessed. Representative dot-plot results for SAMHD1 expression are presented. Percentages indicate absolute SAMHD1 + cell numbers. (b ) Statistical analysis of data obtained as described in a was performed using the Mann-Whitney test. (c) PBMCs were pre-stimulated with IFN-α, or were not stimulated, for 24 h. Cells were then treated for another 48 h with SIV-Vpx or SIV-Mock. Histograms of SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes are presented. (d ) Statistical analysis of data was performed using the Wilcoxon matched pairs test, data from HIV-1 infected individuals (n = 15). ( e ) Purified CD4 + T cells and monocytes were separated from PBMCs, pre-stimulated with PI or IFN-α, then treated with SIV-Vpx or SIV-Mock; whole cell lysates were examined by western blot analysis. Relative protein blot signal intensity analysis was performed using Li-Cor Image Studio software, numbers represent relative value normalized to β-Actin.
    Figure Legend Snippet: The effects of immune activation on SAMHD1 expression and SIV-Vpx mediated SAMHD1 degradation. ( a ) PBMC cells were stimulated with PI (1 μM PMA plus 1 μg/mL Ionomycin) for 24 h. Then, SAMHD1 expression in CD4 + T cells was assessed. Representative dot-plot results for SAMHD1 expression are presented. Percentages indicate absolute SAMHD1 + cell numbers. (b ) Statistical analysis of data obtained as described in a was performed using the Mann-Whitney test. (c) PBMCs were pre-stimulated with IFN-α, or were not stimulated, for 24 h. Cells were then treated for another 48 h with SIV-Vpx or SIV-Mock. Histograms of SAMHD1 expression in activated CD4 + T cells, resting CD4 + T cells, and monocytes are presented. (d ) Statistical analysis of data was performed using the Wilcoxon matched pairs test, data from HIV-1 infected individuals (n = 15). ( e ) Purified CD4 + T cells and monocytes were separated from PBMCs, pre-stimulated with PI or IFN-α, then treated with SIV-Vpx or SIV-Mock; whole cell lysates were examined by western blot analysis. Relative protein blot signal intensity analysis was performed using Li-Cor Image Studio software, numbers represent relative value normalized to β-Actin.

    Techniques Used: Activation Assay, Expressing, MANN-WHITNEY, Infection, Purification, Western Blot, Software

    10) Product Images from "Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network"

    Article Title: Activity of the SPCA1 calcium pump couples sphingomyelin synthesis to sorting of secretory proteins in the trans-Golgi network

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.10.012

    SM depletion inhibits Ca 2+ influx into the TGN. (A) Example time-lapse images of Golgi Ca 2+ influx assays in control, siRNA SMS1/2 or SPCA1 depleted HeLa cells expressing the Go-D1-cpv Golgi Ca 2+ sensor. Fluorescence micrographs of the FRET sensor in the TGN are shown in the left column. Cells were incubated with ionomycin to deplete Ca 2+ from the lumen of the TGN and then live-cell ratiometric FRET microscopy was used to monitor Ca 2+ influx by measuring the ΔR/R 0 FRET ratio of YFP/CFP channels over time after addition of 2.2 mM CaCl 2 to the cell medium, where R 0 is the FRET ratio value obtained before addition of 2.2 mM CaCl 2 (20 second time point). The color-coded ΔR/R 0 heat map scale is shown on the right. Images are shown for representative cells 160 and 300 seconds after addition of CaCl 2 at 80 sec. Scale bars, 5 μm. (B) Quantification of FRET images shown in A, as well as that of siRNA-treated cells expressing siRNA-insensitive SMS1 or SPCA1 cDNAs. Fluorescence signals reflecting TGN [Ca 2+ ] are presented as ΔR/R 0. Data are plotted as the mean Ca 2+ influx over time. Data was acquired for at least 12 cells per condition in two independent experiments. (C) Data shown in B are plotted as the mean ± s.d Ca 2+ influx before Ca 2+ addition (at 80 sec) or after Ca 2+ addition (at 300 sec). Data was acquired for at least 12 cells per condition in two independent experiments. (D) The number of LyzC vesicles was quantified in HeLa control or SMS1/2 siRNA depleted cells expressing either LyzC-SBP-eGFP alone, or co-transfected with SMS1-WT or SPCA1-WT. Vesicle counts from at least 18 cells per condition in 3 independent experiments are plotted. (E) TIRF microscopy-based sorting assays were used to determine the proportion of LyzC-pHluorin exocytic vesicles that also contained EQ-SM-mKate2 in SMS1/2edited cells that express either SPCA1-WT or SPCA1-D350A, a mutant that has no Ca 2+ .
    Figure Legend Snippet: SM depletion inhibits Ca 2+ influx into the TGN. (A) Example time-lapse images of Golgi Ca 2+ influx assays in control, siRNA SMS1/2 or SPCA1 depleted HeLa cells expressing the Go-D1-cpv Golgi Ca 2+ sensor. Fluorescence micrographs of the FRET sensor in the TGN are shown in the left column. Cells were incubated with ionomycin to deplete Ca 2+ from the lumen of the TGN and then live-cell ratiometric FRET microscopy was used to monitor Ca 2+ influx by measuring the ΔR/R 0 FRET ratio of YFP/CFP channels over time after addition of 2.2 mM CaCl 2 to the cell medium, where R 0 is the FRET ratio value obtained before addition of 2.2 mM CaCl 2 (20 second time point). The color-coded ΔR/R 0 heat map scale is shown on the right. Images are shown for representative cells 160 and 300 seconds after addition of CaCl 2 at 80 sec. Scale bars, 5 μm. (B) Quantification of FRET images shown in A, as well as that of siRNA-treated cells expressing siRNA-insensitive SMS1 or SPCA1 cDNAs. Fluorescence signals reflecting TGN [Ca 2+ ] are presented as ΔR/R 0. Data are plotted as the mean Ca 2+ influx over time. Data was acquired for at least 12 cells per condition in two independent experiments. (C) Data shown in B are plotted as the mean ± s.d Ca 2+ influx before Ca 2+ addition (at 80 sec) or after Ca 2+ addition (at 300 sec). Data was acquired for at least 12 cells per condition in two independent experiments. (D) The number of LyzC vesicles was quantified in HeLa control or SMS1/2 siRNA depleted cells expressing either LyzC-SBP-eGFP alone, or co-transfected with SMS1-WT or SPCA1-WT. Vesicle counts from at least 18 cells per condition in 3 independent experiments are plotted. (E) TIRF microscopy-based sorting assays were used to determine the proportion of LyzC-pHluorin exocytic vesicles that also contained EQ-SM-mKate2 in SMS1/2edited cells that express either SPCA1-WT or SPCA1-D350A, a mutant that has no Ca 2+ .

    Techniques Used: Expressing, Fluorescence, Incubation, Microscopy, Size-exclusion Chromatography, Transfection, Mutagenesis

    11) Product Images from "Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress"

    Article Title: Thioesterase-mediated control of cellular calcium homeostasis enables hepatic ER stress

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI93123

    Them2 and PC-TP enable palmitic acid–induced calcium efflux from the ER. ( A – C ) Serum-starved HEK 293E cells were treated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours following knockdown of scrambled control (black/gray) ( A ), Them2 (red/pink) ( B ), or PC-TP (blue/aqua) ( C ). Calcium release into the cytosol was measured as a function of the fluorescence intensity of the cytosolic calcium indicator Fluo4-AM following the induction of ER calcium release by thapsigargin (top panels; Tg, 2 μM) or ionomycin (bottom panels; IO, 5 μM). ( D ) Steady-state cytosolic calcium levels in HEK 293E cells following treatment with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Cells were treated with Them2, PC-TP, or scrambled control siRNA for 48 hours and serum-starved overnight before palmitic acid treatment. * P
    Figure Legend Snippet: Them2 and PC-TP enable palmitic acid–induced calcium efflux from the ER. ( A – C ) Serum-starved HEK 293E cells were treated with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours following knockdown of scrambled control (black/gray) ( A ), Them2 (red/pink) ( B ), or PC-TP (blue/aqua) ( C ). Calcium release into the cytosol was measured as a function of the fluorescence intensity of the cytosolic calcium indicator Fluo4-AM following the induction of ER calcium release by thapsigargin (top panels; Tg, 2 μM) or ionomycin (bottom panels; IO, 5 μM). ( D ) Steady-state cytosolic calcium levels in HEK 293E cells following treatment with palmitic acid (0.5 mM) or vehicle (4.8 mM BSA) for 6 hours. Cells were treated with Them2, PC-TP, or scrambled control siRNA for 48 hours and serum-starved overnight before palmitic acid treatment. * P

    Techniques Used: Fluorescence

    12) Product Images from "Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates"

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

    Journal: Haematologica

    doi: 10.3324/haematol.2018.195057

    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
    Figure Legend 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

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

    13) Product Images from "Oxytocin-Stimulated NFAT Transcriptional Activation in Human Myometrial Cells"

    Article Title: Oxytocin-Stimulated NFAT Transcriptional Activation in Human Myometrial Cells

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2012-1057

    OXT-induced NFATC1-EFP nuclear translocation is inhibited by OXT antagonists, calcineurin inhibitors, and inhibition of Ca 2+ signaling. Human myometrial cells were transduced with a NFATC1-EFP adenovirus before pretreatment with OXT antagonists atosiban (10 −5 m ) and L368899 (10 −5 m ) (A), calcineurin inhibitors CyclA (10 −6 m ) and FK506 (10 −6 m ) (B), and the [Ca 2+ ] i chelator BAPTA-AM (10 −6 m ) and replacement of PSS with Ca 2+ -free solution (C) for 30 min. Cells were stimulated with OXT (10 −7 m ) or ionomycin (2 μ m ) for 60 min before terminating the reaction with ice-cold PBS, fixing in 4% PFA, and staining with Hoechst. Fixed cell image acquisition and analysis were performed as described in Materials and Methods . Data shown are NFATC1-EFP intensity (background subtracted) N:C ratio, mean ± se , from three separate experiments and independent donors. Statistical analyses were performed by one-way ANOVA with Dunnett's post hoc test compared with the untreated control. *, P
    Figure Legend Snippet: OXT-induced NFATC1-EFP nuclear translocation is inhibited by OXT antagonists, calcineurin inhibitors, and inhibition of Ca 2+ signaling. Human myometrial cells were transduced with a NFATC1-EFP adenovirus before pretreatment with OXT antagonists atosiban (10 −5 m ) and L368899 (10 −5 m ) (A), calcineurin inhibitors CyclA (10 −6 m ) and FK506 (10 −6 m ) (B), and the [Ca 2+ ] i chelator BAPTA-AM (10 −6 m ) and replacement of PSS with Ca 2+ -free solution (C) for 30 min. Cells were stimulated with OXT (10 −7 m ) or ionomycin (2 μ m ) for 60 min before terminating the reaction with ice-cold PBS, fixing in 4% PFA, and staining with Hoechst. Fixed cell image acquisition and analysis were performed as described in Materials and Methods . Data shown are NFATC1-EFP intensity (background subtracted) N:C ratio, mean ± se , from three separate experiments and independent donors. Statistical analyses were performed by one-way ANOVA with Dunnett's post hoc test compared with the untreated control. *, P

    Techniques Used: Translocation Assay, Inhibition, Transduction, Staining

    14) Product Images from "Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions"

    Article Title: Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-016-9521-8

    Effect of CGRP on ATP- and ADP-induced Ca 2+ transients in trigeminal neurons. a Intracellular calcium transients evoked by 2 s application of 1 μM ATP, 50 mM KCl, and 10 μM ionomycin in trigeminal neurons in control conditions (average of 5 traces with SEM). b Transients induced by ATP, KCl, and ionomycin after 2 h exposure to 1 μM CGRP (average of 5 traces). c Quantification of CGRP effect on Ca 2+ transients activated by 1 μM ATP ( n = 40 cells) or 1 μM ADP ( n = 40 cells) in control and after CGRP exposure ( n = 84 cells in both cases). Notice that CGRP increased Ca 2+ responses to ATP. Mean ± SEM, * p
    Figure Legend Snippet: Effect of CGRP on ATP- and ADP-induced Ca 2+ transients in trigeminal neurons. a Intracellular calcium transients evoked by 2 s application of 1 μM ATP, 50 mM KCl, and 10 μM ionomycin in trigeminal neurons in control conditions (average of 5 traces with SEM). b Transients induced by ATP, KCl, and ionomycin after 2 h exposure to 1 μM CGRP (average of 5 traces). c Quantification of CGRP effect on Ca 2+ transients activated by 1 μM ATP ( n = 40 cells) or 1 μM ADP ( n = 40 cells) in control and after CGRP exposure ( n = 84 cells in both cases). Notice that CGRP increased Ca 2+ responses to ATP. Mean ± SEM, * p

    Techniques Used:

    15) Product Images from "Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease"

    Article Title: Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease

    Journal: Nature Communications

    doi: 10.1038/ncomms15677

    Mice lacking Ndfip1 in their T reg cells develop inflammatory disease. ( a ) Ndfip1 expression assessed by qPCR before (−) or after (+) αCD3/CD28 or PMA/ionomycin (P/I) stimulation of sorted YFP + T reg cells from WT and Ndfip1 fl/fl Foxp3- Cre mice. A representative example of Ndfip1 expression relative to Actb after αCD3/CD28 stimulation is shown. ( b ) Representative Haematoxylin and Eosin-stained histological sections of the skin, oesophagus and lung from genotypes as indicated are shown. Scale bars represent 100 μM. Far right image in panel is a representative image of the spleen and lymph nodes to illustrate size. ( c ) Inflammation index, calculated as a spleen weight/body weight for male Ndfip1 +/+ Foxp3- Cre (WT) and Ndfip1 fl/fl Foxp3- Cre (cKO) mice at 9–16 weeks of age. ( d ) Levels of serum antibody isotypes as quantified by ELISA. ( e , f ) T cells from lung homogenates were analysed by flow cytometry for ( e ) the percentages of CD44 + cells among CD4 + cells and ( f ) the percentages of CD4 + cells producing the indicated cytokines after ex vivo (P/I) stimulation. P values determined by Student's t -test, with correction for unequal variances as appropriate. * P
    Figure Legend Snippet: Mice lacking Ndfip1 in their T reg cells develop inflammatory disease. ( a ) Ndfip1 expression assessed by qPCR before (−) or after (+) αCD3/CD28 or PMA/ionomycin (P/I) stimulation of sorted YFP + T reg cells from WT and Ndfip1 fl/fl Foxp3- Cre mice. A representative example of Ndfip1 expression relative to Actb after αCD3/CD28 stimulation is shown. ( b ) Representative Haematoxylin and Eosin-stained histological sections of the skin, oesophagus and lung from genotypes as indicated are shown. Scale bars represent 100 μM. Far right image in panel is a representative image of the spleen and lymph nodes to illustrate size. ( c ) Inflammation index, calculated as a spleen weight/body weight for male Ndfip1 +/+ Foxp3- Cre (WT) and Ndfip1 fl/fl Foxp3- Cre (cKO) mice at 9–16 weeks of age. ( d ) Levels of serum antibody isotypes as quantified by ELISA. ( e , f ) T cells from lung homogenates were analysed by flow cytometry for ( e ) the percentages of CD44 + cells among CD4 + cells and ( f ) the percentages of CD4 + cells producing the indicated cytokines after ex vivo (P/I) stimulation. P values determined by Student's t -test, with correction for unequal variances as appropriate. * P

    Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Ex Vivo

    Related Articles

    Flow Cytometry:

    Article Title: Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells
    Article Snippet: .. The cells were then sorted; CD4 enriched; stimulated for 4 h with PMA (30 ng ml−1 , Calbiochem), ionomycin (1 μM, Abcam) and Brefeldin A (1 μg ml−1 , Sigma) for intracellular cytokine staining; or directly stained for flow cytometry. .. Cells were washed in serum-free HBSS or PBS, stained with live/dead fixable blue dead cell stain (L-23105; Invitrogen), Fc blocked (αCD16/32, 2.4G2; BD Biosciences) and stained with the appropriate antibody mixtures.

    Positive Control:

    Article Title: Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates
    Article Snippet: .. Freshly isolated PLTs were incubated with 10 mM ionomycin (Abcam, Cambridge, UK) and used as positive control. ..

    Protease Inhibitor:

    Article Title: Calcium signalling links MYC to NUAK1
    Article Snippet: .. Chemicals and antibodies Phenformin, Sto-609, Rapamycin, phosphatase inhibitor cocktails (P0044 and P5726), protease inhibitor cocktail (P8340) and MG132 were purchased from Sigma-Aldrich (Irvine, UK); HTH-01-015 from Cambridge Bioscience (Cambridge, UK); A23187, Ionomycin and A769662 from Abcam (Cambridge, UK); WZ4003, Gö6976 and BAPTA-AM were purchased from Tocris (Bristol, UK). .. Antibodies recognizing ACC phospho-Ser79(#3661), total ACC (#3676), Raptor phospho-Ser792(#2083), total Raptor (#3661), AMPK phospho-T172(#2535), total AMPK (#2532), total MYPT1 (#8574), PKCα (#2056), NUAK1 (#4458), phospho-(Ser) PKC substrate (#2261), MARCKS phospho-Ser159/163 (#11992) were purchased from Cell Signalling Technologies (Danvers, MA, USA); anti-FLAG (#F1804), anti-β-Actin (#A5441) were from Sigma-Aldrich; anti-MYPT1 phospho-Ser445(#S508C) and anti-NUAK2 (#S225B) were from the MRC PPU, Dundee, UK; anti-MARCKS (#ab72459) anti-Histone H2B (#ab1790), anti-Vinculin (#ab129002) and anti-c-Myc (#ab32072) were purchased from Abcam.

    Transfection:

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: .. Transfected RCMH cells were stimulated with 20 µM ionomycin for 5 min, fixed, immunolabeled with a monoclonal anti-GLUT4 antibody (Abcam, ab35826) and visualized by TIRF microscopy. .. 16-bit raw images were converted to 8-bit, using the ImageJ software and plasma membrane insertion of endogenous GLUT4 was quantified as the total fluorescence intensity of GLUT4 in the evanescent field after background subtraction.

    Microscopy:

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: .. Transfected RCMH cells were stimulated with 20 µM ionomycin for 5 min, fixed, immunolabeled with a monoclonal anti-GLUT4 antibody (Abcam, ab35826) and visualized by TIRF microscopy. .. 16-bit raw images were converted to 8-bit, using the ImageJ software and plasma membrane insertion of endogenous GLUT4 was quantified as the total fluorescence intensity of GLUT4 in the evanescent field after background subtraction.

    Cytometry:

    Article Title: Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells
    Article Snippet: .. The cells were then sorted; CD4 enriched; stimulated for 4 h with PMA (30 ng ml−1 , Calbiochem), ionomycin (1 μM, Abcam) and Brefeldin A (1 μg ml−1 , Sigma) for intracellular cytokine staining; or directly stained for flow cytometry. .. Cells were washed in serum-free HBSS or PBS, stained with live/dead fixable blue dead cell stain (L-23105; Invitrogen), Fc blocked (αCD16/32, 2.4G2; BD Biosciences) and stained with the appropriate antibody mixtures.

    Immunolabeling:

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: .. Transfected RCMH cells were stimulated with 20 µM ionomycin for 5 min, fixed, immunolabeled with a monoclonal anti-GLUT4 antibody (Abcam, ab35826) and visualized by TIRF microscopy. .. 16-bit raw images were converted to 8-bit, using the ImageJ software and plasma membrane insertion of endogenous GLUT4 was quantified as the total fluorescence intensity of GLUT4 in the evanescent field after background subtraction.

    Incubation:

    Article Title: Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates
    Article Snippet: .. Freshly isolated PLTs were incubated with 10 mM ionomycin (Abcam, Cambridge, UK) and used as positive control. ..

    other:

    Article Title: Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling
    Article Snippet: CPA, and ionomycin were purchased from Abcam, Collagenase A was purchased from Roche.

    Blocking Assay:

    Article Title: Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia
    Article Snippet: .. For intracellular cytokine staining of T cells, lung cell suspensions were stimulated with 30 ng/ml phorbol 12-myristate 13-acetate (Millipore, Chicago, IL, USA) and 1 μg/ml ionomycin (Abcam, Boston, MA, USA) in the presence of Brefeldin A (GolgiPlug from BD, New York, NY, USA) for 4 h, then cells were stained with fixable live-dead dye and surface stained for 30 min with the following antibodies in the presence Fc Block: CD4 (clone GK1.5), CD3 (clone 17A2) or TCRb (clone ), and CD8 (clone 53-6.7). .. Cells were then fixed and permeablized with BD Cytofix/Cytoperm kit, (BD, New York, NY, USA) and stained for interferon gamma (IFNγ; clone XMG1.2), IL-4 (clone 11B11) and IL-5 (clone TRFK5) for 1 h. All flow cytometry data was collected at the CHOP flow cytometry core with an LSR Fortessa (BD), and data were analyzed with FlowJo software (Treestar, Ashland, OR, USA).

    Isolation:

    Article Title: Cold storage of platelets in additive solution: the impact of residual plasma in apheresis platelet concentrates
    Article Snippet: .. Freshly isolated PLTs were incubated with 10 mM ionomycin (Abcam, Cambridge, UK) and used as positive control. ..

    Staining:

    Article Title: Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells
    Article Snippet: .. The cells were then sorted; CD4 enriched; stimulated for 4 h with PMA (30 ng ml−1 , Calbiochem), ionomycin (1 μM, Abcam) and Brefeldin A (1 μg ml−1 , Sigma) for intracellular cytokine staining; or directly stained for flow cytometry. .. Cells were washed in serum-free HBSS or PBS, stained with live/dead fixable blue dead cell stain (L-23105; Invitrogen), Fc blocked (αCD16/32, 2.4G2; BD Biosciences) and stained with the appropriate antibody mixtures.

    Article Title: Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia
    Article Snippet: .. For intracellular cytokine staining of T cells, lung cell suspensions were stimulated with 30 ng/ml phorbol 12-myristate 13-acetate (Millipore, Chicago, IL, USA) and 1 μg/ml ionomycin (Abcam, Boston, MA, USA) in the presence of Brefeldin A (GolgiPlug from BD, New York, NY, USA) for 4 h, then cells were stained with fixable live-dead dye and surface stained for 30 min with the following antibodies in the presence Fc Block: CD4 (clone GK1.5), CD3 (clone 17A2) or TCRb (clone ), and CD8 (clone 53-6.7). .. Cells were then fixed and permeablized with BD Cytofix/Cytoperm kit, (BD, New York, NY, USA) and stained for interferon gamma (IFNγ; clone XMG1.2), IL-4 (clone 11B11) and IL-5 (clone TRFK5) for 1 h. All flow cytometry data was collected at the CHOP flow cytometry core with an LSR Fortessa (BD), and data were analyzed with FlowJo software (Treestar, Ashland, OR, USA).

    Software:

    Article Title: Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+
    Article Snippet: .. For Kd determination of MICU1 FRET we used the same buffer containing 3 mM EGTA supplemented with 3–10 μM ionomycin (Abcam Biochemicals, Cambridge, UK) and CaCl2 was added according to the CaBuff software (G. Droogmans, Fysiologie, Leuven) to obtain buffer solutions of 0.1, 1, 10, 100 and 1000 μM free Ca2+ concentrations. .. Cell culture and transfection HeLa cells at passage > 50 were cultured on glass cover slips (Ø = 30 mm) using DMEM (Sigma-Aldrich, Vienna, Austria) containing 10% FCS (PAA, Pasching, Austria), penicillin (100 U/ml) and streptomycin (100 U/ml) in a humidified incubator (37 °C, 5% CO2 /95% air).

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    Abcam ionomycin
    Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in <t>ionomycin</t> permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P
    Ionomycin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 14 article reviews
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    Abcam ionomycin free acid
    Inhibition of mitochondrial proteases increases α-synuclein pathology. A and B, quantification of mitochondrial fragmentation after 5 h treatment with 10 μ m FCCP, 10 μ m BAPTA-AM, 500 μ m MPP + , 1 μ m <t>ionomycin,</t> and 3 μ m menadione. The mitochondrial length was significantly decreased after treatment with FCCP and BAPTA-AM. Scale bars : 10 μ m . Data are presented as mean ± S.D. ****, p
    Ionomycin Free Acid, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin free acid/product/Abcam
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    Image Search Results


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

    Journal: Nitric oxide : biology and chemistry

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

    doi: 10.1016/j.niox.2017.09.001

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

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

    Techniques: Derivative Assay

    Ca 2+ dynamically controls rearrangement of MICU1 multimers in intact cells. ( A ) Schematic illustration of the putative Ca 2+ -induced rearrangement of MICU1-CFP and MICU1-YFP hexamers that bind to the MCU/EMRE mitochondrial Ca 2+ uptake machinery. It is hypothesized that Ca 2+ binding to the MICU1 EF-hands reduces FRET between the respective MICU1-conjugated FPs. ( B ) Representative curve showing the MICU1 FRET ratio signal of HeLa cells co-expressing MICU1-CFP and MICU1-YFP upon cell treatment with 100 μM histamine in the presence of 2 mM Ca 2+ . ( C ) Representative images to panel b under basal condition, upon stimulation with 100 μM histamine 3 min. after stimulation (scale bar, 10 μm). ( D ) Representative MICU1 FRET ratio signals over time in HeLa cells upon addition and removal of different free Ca 2+ concentrations in the presence of 3 μM ionomycin. The maximal ∆ MICU1 FRET ratio signal (between 3 mM EGTA and 1000 μM Ca 2+ ) was defined as 100%. ( E ) Concentration response curve of the Ca 2+ -induced reduction of the MICU1 FRET ratio signal in HeLa cells that were treated with 3 μM ionomycin and different Ca 2+ concentrations as shown in panel ( D ); mean ± SEM. (n = 7–9).

    Journal: Scientific Reports

    Article Title: Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+

    doi: 10.1038/srep15602

    Figure Lengend Snippet: Ca 2+ dynamically controls rearrangement of MICU1 multimers in intact cells. ( A ) Schematic illustration of the putative Ca 2+ -induced rearrangement of MICU1-CFP and MICU1-YFP hexamers that bind to the MCU/EMRE mitochondrial Ca 2+ uptake machinery. It is hypothesized that Ca 2+ binding to the MICU1 EF-hands reduces FRET between the respective MICU1-conjugated FPs. ( B ) Representative curve showing the MICU1 FRET ratio signal of HeLa cells co-expressing MICU1-CFP and MICU1-YFP upon cell treatment with 100 μM histamine in the presence of 2 mM Ca 2+ . ( C ) Representative images to panel b under basal condition, upon stimulation with 100 μM histamine 3 min. after stimulation (scale bar, 10 μm). ( D ) Representative MICU1 FRET ratio signals over time in HeLa cells upon addition and removal of different free Ca 2+ concentrations in the presence of 3 μM ionomycin. The maximal ∆ MICU1 FRET ratio signal (between 3 mM EGTA and 1000 μM Ca 2+ ) was defined as 100%. ( E ) Concentration response curve of the Ca 2+ -induced reduction of the MICU1 FRET ratio signal in HeLa cells that were treated with 3 μM ionomycin and different Ca 2+ concentrations as shown in panel ( D ); mean ± SEM. (n = 7–9).

    Article Snippet: For Kd determination of MICU1 FRET we used the same buffer containing 3 mM EGTA supplemented with 3–10 μM ionomycin (Abcam Biochemicals, Cambridge, UK) and CaCl2 was added according to the CaBuff software (G. Droogmans, Fysiologie, Leuven) to obtain buffer solutions of 0.1, 1, 10, 100 and 1000 μM free Ca2+ concentrations.

    Techniques: Binding Assay, Expressing, Concentration Assay

    The Ca 2+ -induced rearrangement of MICU1 multimers is independent of the expression level of MCU and EMRE. ( A ) Average curves of MICU1 FRET ratio signals over time in response to 100 μM histamine in Ca 2+ -free solution of control HeLa cells (black curve, n = 23) and cells treated with siRNA against MCU (blue curve, n = 22). ( B ) Bars represent maximal ∆ MICU1 FRET ratio values (mean ± SEM) extracted from curves shown in panel A. ( C ) Bars represent maximal ∆ MICU1 FRET ratio values upon cell treatment with 100 μM histamine in Ca 2+ -free solution of control HeLa cells (white column, n = 19) and cells treated with siRNA against EMRE (red column, n = 12). ( D ) Concentration response curves showing the effects of different Ca 2+ concentrations on the MICU1 FRET ratio in ionomycin- (3 μM) treated control HeLa cells (black curve, white circles, n = 7–9), cells reduced of MCU (blue dotted curve, blue filled squares, n = 8–10), and cells reduced of EMRE (red dotted curve, r ed filled rhombs, n = 10).

    Journal: Scientific Reports

    Article Title: Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+

    doi: 10.1038/srep15602

    Figure Lengend Snippet: The Ca 2+ -induced rearrangement of MICU1 multimers is independent of the expression level of MCU and EMRE. ( A ) Average curves of MICU1 FRET ratio signals over time in response to 100 μM histamine in Ca 2+ -free solution of control HeLa cells (black curve, n = 23) and cells treated with siRNA against MCU (blue curve, n = 22). ( B ) Bars represent maximal ∆ MICU1 FRET ratio values (mean ± SEM) extracted from curves shown in panel A. ( C ) Bars represent maximal ∆ MICU1 FRET ratio values upon cell treatment with 100 μM histamine in Ca 2+ -free solution of control HeLa cells (white column, n = 19) and cells treated with siRNA against EMRE (red column, n = 12). ( D ) Concentration response curves showing the effects of different Ca 2+ concentrations on the MICU1 FRET ratio in ionomycin- (3 μM) treated control HeLa cells (black curve, white circles, n = 7–9), cells reduced of MCU (blue dotted curve, blue filled squares, n = 8–10), and cells reduced of EMRE (red dotted curve, r ed filled rhombs, n = 10).

    Article Snippet: For Kd determination of MICU1 FRET we used the same buffer containing 3 mM EGTA supplemented with 3–10 μM ionomycin (Abcam Biochemicals, Cambridge, UK) and CaCl2 was added according to the CaBuff software (G. Droogmans, Fysiologie, Leuven) to obtain buffer solutions of 0.1, 1, 10, 100 and 1000 μM free Ca2+ concentrations.

    Techniques: Expressing, Concentration Assay

    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

    Inhibition of mitochondrial proteases increases α-synuclein pathology. A and B, quantification of mitochondrial fragmentation after 5 h treatment with 10 μ m FCCP, 10 μ m BAPTA-AM, 500 μ m MPP + , 1 μ m ionomycin, and 3 μ m menadione. The mitochondrial length was significantly decreased after treatment with FCCP and BAPTA-AM. Scale bars : 10 μ m . Data are presented as mean ± S.D. ****, p

    Journal: The Journal of Biological Chemistry

    Article Title: Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies

    doi: 10.1074/jbc.RA119.011650

    Figure Lengend Snippet: Inhibition of mitochondrial proteases increases α-synuclein pathology. A and B, quantification of mitochondrial fragmentation after 5 h treatment with 10 μ m FCCP, 10 μ m BAPTA-AM, 500 μ m MPP + , 1 μ m ionomycin, and 3 μ m menadione. The mitochondrial length was significantly decreased after treatment with FCCP and BAPTA-AM. Scale bars : 10 μ m . Data are presented as mean ± S.D. ****, p

    Article Snippet: FCCP ( Abcam, Cambridge, UK), 1 mm in DMSO, MPP+ ( Sigma-Aldrich), 10 mm in water, ionomycin (ab120370, Abcam) 10 mm , and 1 mm in DMSO, 2-deoxyglucose (Sigma-Aldrich) 0.5 m in water, menadione (Sigma-Aldrich), 1.5 mm in DMSO, BAPTA-AM (ab120503, Abcam), 2.5 mm in DMSO, BAPTA (ab144924, Abcam), 1 mm in water, CDDO-Me (Sigma-Aldrich), 1 mm in DMSO, UCF-101 (Sigma-Aldrich), 10 mm in DMSO, MitobloCK-6 (Focus Biomolecules), 5 mm in DMSO, bafilomycin A1 (Calbiochem, San Diego, CA, USA), 100 and 10 μm in DMSO, 17-AAG (ab141433, Abcam), 5 mm and 50 μm in DMSO and MG132 (Sigma-Aldrich), and 10 and 1 mm in DMSO.

    Techniques: Inhibition

    Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p

    Journal: The Journal of Biological Chemistry

    Article Title: Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies

    doi: 10.1074/jbc.RA119.011650

    Figure Lengend Snippet: Downstream effectors of mitochondrial dysfunction do not influence α-synuclein pathology. A, YFP–α-synuclein SH-SY5Y cells were treated with DMSO (control), 500 μ m MPP + , 1 μ m ionomycin, or 3 μ m menadione for 3 days (1 h before, during α-synuclein fibrillar seed incubation, and during the 3-day period until evaluation). Scale bars : 20 μ m . α-Synuclein seeding was not significantly increased (one-way ANOVA with Dunnett's post hoc correction). Data are presented as mean ± S.D., n = 11, 8, 8, and 7 with n = regions analyzed, three biological repeats. B, fluorescence lifetime images and graphs for ATP levels (Ateam1.03 donor fluorescence lifetime) in SH-SY5Y cells treated with DMSO (control), 500 μ m MPP + , 10 μ m FCCP, and 10 μ m BAPTA-AM for 1 h. MPP + and FCCP significantly decreased ATP levels, BAPTA-AM had no significant effect. ****, p

    Article Snippet: FCCP ( Abcam, Cambridge, UK), 1 mm in DMSO, MPP+ ( Sigma-Aldrich), 10 mm in water, ionomycin (ab120370, Abcam) 10 mm , and 1 mm in DMSO, 2-deoxyglucose (Sigma-Aldrich) 0.5 m in water, menadione (Sigma-Aldrich), 1.5 mm in DMSO, BAPTA-AM (ab120503, Abcam), 2.5 mm in DMSO, BAPTA (ab144924, Abcam), 1 mm in water, CDDO-Me (Sigma-Aldrich), 1 mm in DMSO, UCF-101 (Sigma-Aldrich), 10 mm in DMSO, MitobloCK-6 (Focus Biomolecules), 5 mm in DMSO, bafilomycin A1 (Calbiochem, San Diego, CA, USA), 100 and 10 μm in DMSO, 17-AAG (ab141433, Abcam), 5 mm and 50 μm in DMSO and MG132 (Sigma-Aldrich), and 10 and 1 mm in DMSO.

    Techniques: Incubation, Fluorescence