Structured Review

LC Laboratories ionomycin
LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM <t>ionomycin.</t> D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P
Ionomycin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 1 article reviews
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92/100 stars

Images

1) Product Images from "NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx"

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

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00298.2015

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

Techniques Used: Fluorescence, Incubation

2) Product Images from "Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore"

Article Title: Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

Journal: Biology Open

doi: 10.1242/bio.035287

Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P
Figure Legend Snippet: Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

Techniques Used: Microscopy, Expressing, Derivative Assay, Imaging

3) Product Images from "mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling"

Article Title: mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0061020

Rapamycin affects intracellular Ca 2+ signaling. A) Representative measurements ( n = 4) of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca 2+ as indicated. B) Quantification of the average amplitude of the response (F−F 0 ) ( n = 4). * p
Figure Legend Snippet: Rapamycin affects intracellular Ca 2+ signaling. A) Representative measurements ( n = 4) of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca 2+ as indicated. B) Quantification of the average amplitude of the response (F−F 0 ) ( n = 4). * p

Techniques Used:

Changes in Ca 2+ signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex. A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h ( n = 3). B–C) Representative measurements of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca 2+ as indicated. D) Quantification of the average amplitude of the response (F−F 0 ) ( n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p
Figure Legend Snippet: Changes in Ca 2+ signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex. A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h ( n = 3). B–C) Representative measurements of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca 2+ as indicated. D) Quantification of the average amplitude of the response (F−F 0 ) ( n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p

Techniques Used: Western Blot

4) Product Images from "Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore"

Article Title: Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

Journal: Biology Open

doi: 10.1242/bio.035287

Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P
Figure Legend Snippet: Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

Techniques Used: Microscopy, Expressing, Derivative Assay, Imaging

5) Product Images from "Mucosal Immune Response to Feline Enteric Coronavirus Infection"

Article Title: Mucosal Immune Response to Feline Enteric Coronavirus Infection

Journal: Viruses

doi: 10.3390/v11100906

Infection by feline enteric coronavirus (FECV) did not result in an increase in total IFNγ-producing cells or FCoV-specific IFNγ-producing cells. The total number of IFNγ-secreting cells after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation (spot forming units, SFU) ( a ) and the number of FCoV-specific IFNγ-secreting cells ( b ) were determined by ELISPOT. Each symbol represents a single cat. Bars show the mean and 95% confidence interval. Linear models were used to compare group means, and p values are shown when ≤0.05. One was added to the number of FCoV-specific IFNγ-secreting cells prior to log transformation.
Figure Legend Snippet: Infection by feline enteric coronavirus (FECV) did not result in an increase in total IFNγ-producing cells or FCoV-specific IFNγ-producing cells. The total number of IFNγ-secreting cells after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation (spot forming units, SFU) ( a ) and the number of FCoV-specific IFNγ-secreting cells ( b ) were determined by ELISPOT. Each symbol represents a single cat. Bars show the mean and 95% confidence interval. Linear models were used to compare group means, and p values are shown when ≤0.05. One was added to the number of FCoV-specific IFNγ-secreting cells prior to log transformation.

Techniques Used: Infection, Enzyme-linked Immunospot, Transformation Assay

6) Product Images from "HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.15077

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).
Figure Legend 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).

Techniques Used: 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).
Figure Legend 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).

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

7) Product Images from "Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated"

Article Title: Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

Journal: Autophagy

doi: 10.4161/auto.7.12.17909

Transient changes in Ca 2+ dynamics after starvation correlate with changes of the LC3-II levels. (A) Measurements of cytosolic Ca 2+ signals, displayed as normalized Fura-2 ratio (R/R 0 ), showing the effect of 1 µM thapsigargin (TG) or 10 µM ionomycin in intact HeLa cells with (2, 3, 5 h) or without (0 h) starvation. Representative recordings of the Fura-2 ratio after addition of TG (left panel) or ionomycin (right panel) in cells pretreated with HBSS for 0, 2, 3 or 5 h are shown. Forty-five seconds prior to addition of TG or ionomycin, EGTA (3 mM) was given as indicated. (B) Measurements of cytosolic Ca 2+ signals, displayed as normalized Fura-2 ratio (R/R 0 ), after addition of 0.3 µM (left panel) or 100 µM (right panel) ATP in intact HeLa cells with (2, 3, 5 h) or without (0 h) starvation. (C) Representative LC3-II western blots of HeLa cells starved for the indicated time period (0–7 h). (D) GFP-LC3-puncta formation in HeLa cells transfected with GFP-LC3 constructs. Cells were starved (3 h or 5 h) or not (0 h), as indicated. Left: Representative pictures. The scale bar represents 20 µm. Right: Quantification of autophagic cells. Only cells displaying more than 10 puncta were considered autophagic (n = 3). (E) Analysis of XBP-1-mRNA splicing in cells pretreated with HBSS (0–5 h) or 2 µg/ml tunicamycin (TM; 24 h, 48 h); uXBP-1 = unspliced XBP-1; sXBP-1 = spliced XBP-1. (F) Representative caspase 3 western blots of HeLa cells starved for the indicated time (0–5 h) or treated with 1 µM staurosporine (STS) for 5 h. (G) XTT-assay for measurement of cell viability, after incubation in HBSS for the indicated time period or treatment with 1 µM staurosporine for 5 h (n = 4). The cell viability is expressed as the absorbance at 490 nm minus the absorbance at 630 nm (A 490 -A 630 ). *p
Figure Legend Snippet: Transient changes in Ca 2+ dynamics after starvation correlate with changes of the LC3-II levels. (A) Measurements of cytosolic Ca 2+ signals, displayed as normalized Fura-2 ratio (R/R 0 ), showing the effect of 1 µM thapsigargin (TG) or 10 µM ionomycin in intact HeLa cells with (2, 3, 5 h) or without (0 h) starvation. Representative recordings of the Fura-2 ratio after addition of TG (left panel) or ionomycin (right panel) in cells pretreated with HBSS for 0, 2, 3 or 5 h are shown. Forty-five seconds prior to addition of TG or ionomycin, EGTA (3 mM) was given as indicated. (B) Measurements of cytosolic Ca 2+ signals, displayed as normalized Fura-2 ratio (R/R 0 ), after addition of 0.3 µM (left panel) or 100 µM (right panel) ATP in intact HeLa cells with (2, 3, 5 h) or without (0 h) starvation. (C) Representative LC3-II western blots of HeLa cells starved for the indicated time period (0–7 h). (D) GFP-LC3-puncta formation in HeLa cells transfected with GFP-LC3 constructs. Cells were starved (3 h or 5 h) or not (0 h), as indicated. Left: Representative pictures. The scale bar represents 20 µm. Right: Quantification of autophagic cells. Only cells displaying more than 10 puncta were considered autophagic (n = 3). (E) Analysis of XBP-1-mRNA splicing in cells pretreated with HBSS (0–5 h) or 2 µg/ml tunicamycin (TM; 24 h, 48 h); uXBP-1 = unspliced XBP-1; sXBP-1 = spliced XBP-1. (F) Representative caspase 3 western blots of HeLa cells starved for the indicated time (0–5 h) or treated with 1 µM staurosporine (STS) for 5 h. (G) XTT-assay for measurement of cell viability, after incubation in HBSS for the indicated time period or treatment with 1 µM staurosporine for 5 h (n = 4). The cell viability is expressed as the absorbance at 490 nm minus the absorbance at 630 nm (A 490 -A 630 ). *p

Techniques Used: Western Blot, Transfection, Construct, XTT Assay, Incubation

Related Articles

Enzyme-linked Immunospot:

Article Title: Mucosal Immune Response to Feline Enteric Coronavirus Infection
Article Snippet: .. IFNγ ELISPOT Capture and detection antibodies from the feline IFNγ Development Module (R & D Systems) were used with MultiScreen-IP 96-well plates (MAIPSWU10; MilliporeSigma) to quantify IFNγ-producing mucosal lymphocytes after stimulation with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; LC Laboratories, Woburn, MA) and ionomycin (300 ng/mL; LC Laboratories) or with FIPV antigen (60 µg/mL; IVD Technologies, Santa Ana, CA, USA) as previously described [ ]. .. The protocol was modified with the use of 2.5 × 104 cells/well (PMA/ionomycin) or 2 × 105 cells/well (FIPV antigen) incubated for 40 h at 37 °C, 5% CO2 .

In Vitro:

Article Title: HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function
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). .. 1.5 hours later cells were cultured for an additional 2.5 h in the presence of Protein Transport Inhibitor Brefeldin A (1μl/ml, eBioscience).

Cell Culture:

Article Title: HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function
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). .. 1.5 hours later cells were cultured for an additional 2.5 h in the presence of Protein Transport Inhibitor Brefeldin A (1μl/ml, eBioscience).

Cell Stimulation:

Article Title: HDAC4 is expressed on multiple T cell lineages but dispensable for their development and function
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). .. 1.5 hours later cells were cultured for an additional 2.5 h in the presence of Protein Transport Inhibitor Brefeldin A (1μl/ml, eBioscience).

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    LC Laboratories ionomycin
    LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM <t>ionomycin.</t> D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P
    Ionomycin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/LC Laboratories
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

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

    Journal: American Journal of Physiology - Cell Physiology

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

    doi: 10.1152/ajpcell.00298.2015

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

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

    Techniques: Fluorescence, Incubation

    Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Journal: Biology Open

    Article Title: Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

    doi: 10.1242/bio.035287

    Figure Lengend Snippet: Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Article Snippet: FM4-64FX and Fluo-3 were from Molecular Probes, Ionomycin from LC Laboratories and Bapta-AM from Tocris Bioscience.

    Techniques: Microscopy, Expressing, Derivative Assay, Imaging

    Rapamycin affects intracellular Ca 2+ signaling. A) Representative measurements ( n = 4) of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca 2+ as indicated. B) Quantification of the average amplitude of the response (F−F 0 ) ( n = 4). * p

    Journal: PLoS ONE

    Article Title: mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

    doi: 10.1371/journal.pone.0061020

    Figure Lengend Snippet: Rapamycin affects intracellular Ca 2+ signaling. A) Representative measurements ( n = 4) of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca 2+ as indicated. B) Quantification of the average amplitude of the response (F−F 0 ) ( n = 4). * p

    Article Snippet: The chemicals used were: A23187 and IP3 (Sigma-Aldrich NV), EGTA (Acros Organics BVBA, Geel, Belgium), thapsigargin (Enzo Life Sciences BVBA, Antwerp, Belgium), ionomycin, rapamycin and bafilomycin A1 (LC laboratories, Woburn, MA), ATP (Roche Diagnostics, Vilvoorde, Belgium), 45 Ca2+ (PerkinElmer, Zaventem, Belgium), Fura2-AM (Biotium, Hayward, CA), and BAPTA-AM (Molecular Probes, Life Technologies).

    Techniques:

    Changes in Ca 2+ signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex. A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h ( n = 3). B–C) Representative measurements of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca 2+ as indicated. D) Quantification of the average amplitude of the response (F−F 0 ) ( n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p

    Journal: PLoS ONE

    Article Title: mTOR-Controlled Autophagy Requires Intracellular Ca2+ Signaling

    doi: 10.1371/journal.pone.0061020

    Figure Lengend Snippet: Changes in Ca 2+ signaling are independent of autophagy stimulation and occur upstream of the Atg12-Atg5 complex. A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h ( n = 3). B–C) Representative measurements of cytosolic Ca 2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca 2+ as indicated. D) Quantification of the average amplitude of the response (F−F 0 ) ( n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p

    Article Snippet: The chemicals used were: A23187 and IP3 (Sigma-Aldrich NV), EGTA (Acros Organics BVBA, Geel, Belgium), thapsigargin (Enzo Life Sciences BVBA, Antwerp, Belgium), ionomycin, rapamycin and bafilomycin A1 (LC laboratories, Woburn, MA), ATP (Roche Diagnostics, Vilvoorde, Belgium), 45 Ca2+ (PerkinElmer, Zaventem, Belgium), Fura2-AM (Biotium, Hayward, CA), and BAPTA-AM (Molecular Probes, Life Technologies).

    Techniques: Western Blot

    Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Journal: Biology Open

    Article Title: Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

    doi: 10.1242/bio.035287

    Figure Lengend Snippet: Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Article Snippet: FM4-64FX and Fluo-3 were from Molecular Probes, Ionomycin from LC Laboratories and Bapta-AM from Tocris Bioscience.

    Techniques: Microscopy, Expressing, Derivative Assay, Imaging