atp  (Thermo Fisher)


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    Name:
    ATP Solution
    Description:
    This Ambion ribonucleotide triphosphate rNTP solution is a component of the MAXIscript Kits SKU s AM1312 AM1316 AM1314 AM1326 AM1324 AM1318 AM1322 AM1320 AM1310 and AM1308 and is supplied in one tube containing 100 μL Each is subjected to RNase testing as well as a functional assay to ensure high levels of incorporation It may also be used in ligation and phosphorylation reactions
    Catalog Number:
    am8110g
    Price:
    None
    Applications:
    In Vitro Transcription|PCR & Real-Time PCR|Gene Expression Analysis & Genotyping
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher atp
    Calcium transients induce nuclear F-actin assembly. a Representative images of NIH3T3 cells stably expressing nAC-GFP after <t>A23187</t> stimulation. In total, 73.6% ± 7.6% (s.e.m.) cells exhibited NAA. Each experiment included three samples in parallel, and about 30 cells were recorded and counted each time. Experiments were repeated three times ( n = 3). Scale bar: 10 μm. b NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum or A23187 (750 nM) after pre-treatment with or without LPA receptor inhibitor Ki16425 (20 μM) at the confocal microscope. n = 4 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. c NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum after pre-treatment with or without BAPTA-AM (10 μM). n = 3 independent experiments. Two-sided Student's t test, ** p ≤ 0.01. d NIH3T3 cells stably expressing nAC-GFP were stimulated with LPA (20 μM), thrombin (0.2 U/mL), or <t>ATP</t> (10 μM). n = 3 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. Error bars: +s.e.m. e Representative images of NIH3T3 cells stably expressing nAC-GFP after thrombin stimulation. Experiments were performed three times, and about 30 cells were recorded each time. Eighty percent of the cells were positive for NAA as shown in panel d . Scale bar: 10 μm. Source data are provided as a Source Data file
    This Ambion ribonucleotide triphosphate rNTP solution is a component of the MAXIscript Kits SKU s AM1312 AM1316 AM1314 AM1326 AM1324 AM1318 AM1322 AM1320 AM1310 and AM1308 and is supplied in one tube containing 100 μL Each is subjected to RNase testing as well as a functional assay to ensure high levels of incorporation It may also be used in ligation and phosphorylation reactions
    https://www.bioz.com/result/atp/product/Thermo Fisher
    Average 99 stars, based on 64 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics"

    Article Title: GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13322-y

    Calcium transients induce nuclear F-actin assembly. a Representative images of NIH3T3 cells stably expressing nAC-GFP after A23187 stimulation. In total, 73.6% ± 7.6% (s.e.m.) cells exhibited NAA. Each experiment included three samples in parallel, and about 30 cells were recorded and counted each time. Experiments were repeated three times ( n = 3). Scale bar: 10 μm. b NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum or A23187 (750 nM) after pre-treatment with or without LPA receptor inhibitor Ki16425 (20 μM) at the confocal microscope. n = 4 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. c NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum after pre-treatment with or without BAPTA-AM (10 μM). n = 3 independent experiments. Two-sided Student's t test, ** p ≤ 0.01. d NIH3T3 cells stably expressing nAC-GFP were stimulated with LPA (20 μM), thrombin (0.2 U/mL), or ATP (10 μM). n = 3 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. Error bars: +s.e.m. e Representative images of NIH3T3 cells stably expressing nAC-GFP after thrombin stimulation. Experiments were performed three times, and about 30 cells were recorded each time. Eighty percent of the cells were positive for NAA as shown in panel d . Scale bar: 10 μm. Source data are provided as a Source Data file
    Figure Legend Snippet: Calcium transients induce nuclear F-actin assembly. a Representative images of NIH3T3 cells stably expressing nAC-GFP after A23187 stimulation. In total, 73.6% ± 7.6% (s.e.m.) cells exhibited NAA. Each experiment included three samples in parallel, and about 30 cells were recorded and counted each time. Experiments were repeated three times ( n = 3). Scale bar: 10 μm. b NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum or A23187 (750 nM) after pre-treatment with or without LPA receptor inhibitor Ki16425 (20 μM) at the confocal microscope. n = 4 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. c NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum after pre-treatment with or without BAPTA-AM (10 μM). n = 3 independent experiments. Two-sided Student's t test, ** p ≤ 0.01. d NIH3T3 cells stably expressing nAC-GFP were stimulated with LPA (20 μM), thrombin (0.2 U/mL), or ATP (10 μM). n = 3 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. Error bars: +s.e.m. e Representative images of NIH3T3 cells stably expressing nAC-GFP after thrombin stimulation. Experiments were performed three times, and about 30 cells were recorded each time. Eighty percent of the cells were positive for NAA as shown in panel d . Scale bar: 10 μm. Source data are provided as a Source Data file

    Techniques Used: Stable Transfection, Expressing, Microscopy

    2) Product Images from "Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes"

    Article Title: Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168328

    HBV/HBx requires extracellular Ca 2+ influx to elevate [Ca 2+ ] c . (A) Primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ) or in Ca 2+ -containing buffer (2 mM Ca 2+ ). (B and C) Control and HBx-expressing primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ). Calculations were performed as described for Fig 1 . The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. **P
    Figure Legend Snippet: HBV/HBx requires extracellular Ca 2+ influx to elevate [Ca 2+ ] c . (A) Primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ) or in Ca 2+ -containing buffer (2 mM Ca 2+ ). (B and C) Control and HBx-expressing primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ). Calculations were performed as described for Fig 1 . The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. **P

    Techniques Used: Expressing

    HBV/HBx increases [Ca 2+ ] c in primary rat hepatocytes. (A) RT-qPCR was performed on freshly isolated hepatocytes (0 hr) and on hepatocytes which had been plated for 48 hours (48 hr) for the hepatocyte-specific markers albumin, HNF4α, and transferrin. (B, C, and D) Primary rat hepatocytes were transfected with a control (pGEM) or HBV-expressing vector. 24 hrs post transfection, HBcAg expression was confirmed via western blot (B), and control and HBV-transfected cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (C) or 100 nM Vasopressin (Vp) (D). (E and F) Primary rat hepatocytes were transfected with an HBV-expressing or HBV(HBx-deficient) (HBV*7)-expressing vector. 24 hrs post transfection, equal levels of HBcAg expression were confirmed via western blot (E), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (F). (G and H) Primary rat hepatocytes were transfected with a control (pcDNA) or HBx-expressing vector. 24 hrs post transfection, HBx expression was confirmed via western blot (G), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (H). For all Ca 2+ -imaging experiments, the peak [Ca 2+ ] c was calculated as the change in the peak Fura-4F ratio and the basal Fura-4F ratio (R peak −R basal ). The plateau [Ca 2+ ] c was calculated as a percentage of the peak [(R plateau −R basal ) / (R peak −R basal )]. The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. *P
    Figure Legend Snippet: HBV/HBx increases [Ca 2+ ] c in primary rat hepatocytes. (A) RT-qPCR was performed on freshly isolated hepatocytes (0 hr) and on hepatocytes which had been plated for 48 hours (48 hr) for the hepatocyte-specific markers albumin, HNF4α, and transferrin. (B, C, and D) Primary rat hepatocytes were transfected with a control (pGEM) or HBV-expressing vector. 24 hrs post transfection, HBcAg expression was confirmed via western blot (B), and control and HBV-transfected cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (C) or 100 nM Vasopressin (Vp) (D). (E and F) Primary rat hepatocytes were transfected with an HBV-expressing or HBV(HBx-deficient) (HBV*7)-expressing vector. 24 hrs post transfection, equal levels of HBcAg expression were confirmed via western blot (E), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (F). (G and H) Primary rat hepatocytes were transfected with a control (pcDNA) or HBx-expressing vector. 24 hrs post transfection, HBx expression was confirmed via western blot (G), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (H). For all Ca 2+ -imaging experiments, the peak [Ca 2+ ] c was calculated as the change in the peak Fura-4F ratio and the basal Fura-4F ratio (R peak −R basal ). The plateau [Ca 2+ ] c was calculated as a percentage of the peak [(R plateau −R basal ) / (R peak −R basal )]. The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. *P

    Techniques Used: Quantitative RT-PCR, Isolation, Transfection, Expressing, Plasmid Preparation, Western Blot, Imaging

    3) Product Images from "Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism"

    Article Title: Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54693-y

    Oocytes utilise AMP for ATP production via the adenosine salvage pathway. Iodoacetamide (IAC) was used to inhibit creatine kinase activity in order to perturb the interconversion of ADP and ATP. COCs ( A ) or denuded oocytes ( B ) were cultured for 3 h ± 13 C 5 -AMP and then intra-oocyte 13 C 5 -AMP, 13 C 5 -ADP and 13 C 5 -ATP were measured by LC-MS/MS (n = 4 biological replicates). COCs were cultured with 13 C 5 -AMP ± the gap-junction uncoupler carbenoxolone (CBX) for 3 h and intra-oocyte 13 C 5 -ATP was measured (n = 4 biological replicates) ( C ). COCs were cultured for 3 h with increasing concentrations of 13 C 5 -AMP and the peak areas of intra-oocyte 13 C 5 -ATP and endogenous ATP were measured (n = 1 biological replicate) ( D ). COCs were cultured for 3 h ± 13 C 5 -AMP (1 mM) and intra-oocyte 13 C 5 -ATP was measured (n = 4 biological replicates) ( E ). ns, not significantly different (P ˃ 0.05, t-test), # Not detected; data are mean ± SEM.
    Figure Legend Snippet: Oocytes utilise AMP for ATP production via the adenosine salvage pathway. Iodoacetamide (IAC) was used to inhibit creatine kinase activity in order to perturb the interconversion of ADP and ATP. COCs ( A ) or denuded oocytes ( B ) were cultured for 3 h ± 13 C 5 -AMP and then intra-oocyte 13 C 5 -AMP, 13 C 5 -ADP and 13 C 5 -ATP were measured by LC-MS/MS (n = 4 biological replicates). COCs were cultured with 13 C 5 -AMP ± the gap-junction uncoupler carbenoxolone (CBX) for 3 h and intra-oocyte 13 C 5 -ATP was measured (n = 4 biological replicates) ( C ). COCs were cultured for 3 h with increasing concentrations of 13 C 5 -AMP and the peak areas of intra-oocyte 13 C 5 -ATP and endogenous ATP were measured (n = 1 biological replicate) ( D ). COCs were cultured for 3 h ± 13 C 5 -AMP (1 mM) and intra-oocyte 13 C 5 -ATP was measured (n = 4 biological replicates) ( E ). ns, not significantly different (P ˃ 0.05, t-test), # Not detected; data are mean ± SEM.

    Techniques Used: Activity Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Hypothesised model of the impact of cAMP modulation in the cumulus-oocyte complex on oocyte energy production. Oocyte meiotic resumption and pharmacological cAMP upregulation via pre-IVM generate AMP from cAMP in cumulus cells which can be used via the adenosine salvage pathway to regenerate ATP in the oocyte. Upregulation of cAMP in the cumulus-oocyte complex leads to a decrease in intra-oocyte ATP, decreasing the ATP:ADP ratio and upregulating AMPK protein expression to restrain energy depletion. Pre-IVM also increases COC ATP by stimulating cumulus cell glycolysis. TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AMPK, AMP-activated protein kinase.
    Figure Legend Snippet: Hypothesised model of the impact of cAMP modulation in the cumulus-oocyte complex on oocyte energy production. Oocyte meiotic resumption and pharmacological cAMP upregulation via pre-IVM generate AMP from cAMP in cumulus cells which can be used via the adenosine salvage pathway to regenerate ATP in the oocyte. Upregulation of cAMP in the cumulus-oocyte complex leads to a decrease in intra-oocyte ATP, decreasing the ATP:ADP ratio and upregulating AMPK protein expression to restrain energy depletion. Pre-IVM also increases COC ATP by stimulating cumulus cell glycolysis. TCA, tricarboxylic acid cycle; OXPHOS, oxidative phosphorylation; AMPK, AMP-activated protein kinase.

    Techniques Used: Expressing

    Elevated COC cAMP alters intra-oocyte adenine nucleotide levels throughout oocyte maturation. COCs were either not exposed (control, 0 h pre-IVM) or were exposed to 2 h of pre-IVM with FSK + IBMX prior to IVM culture in the presence of FSH for up to 16 h. Cumulus cells were then removed and intra-oocyte ATP ( A ), ADP ( B ), and the ATP:ADP ratio ( C ) were measured (mean ± SEM). T, treatment; D, oocyte culture duration; *Significantly different (P
    Figure Legend Snippet: Elevated COC cAMP alters intra-oocyte adenine nucleotide levels throughout oocyte maturation. COCs were either not exposed (control, 0 h pre-IVM) or were exposed to 2 h of pre-IVM with FSK + IBMX prior to IVM culture in the presence of FSH for up to 16 h. Cumulus cells were then removed and intra-oocyte ATP ( A ), ADP ( B ), and the ATP:ADP ratio ( C ) were measured (mean ± SEM). T, treatment; D, oocyte culture duration; *Significantly different (P

    Techniques Used:

    Elevated COC cAMP alters adenine nucleotide levels. COCs were either untreated (control) or pre-treated for 2 h (pre-IVM) with FSK + IBMX, then cultured without treatments in the presence of FSH for 3 h. COC ATP ( A ), ADP ( B ), AMP ( C ) were measured. COC ATP:ADP ( D ), ATP:AMP ( E ) ratios and energy charge ( F ) were calculated. N = 4 biological replicates, data are mean ± SEM. T, treatment; D, oocyte culture duration; *Significantly different (P
    Figure Legend Snippet: Elevated COC cAMP alters adenine nucleotide levels. COCs were either untreated (control) or pre-treated for 2 h (pre-IVM) with FSK + IBMX, then cultured without treatments in the presence of FSH for 3 h. COC ATP ( A ), ADP ( B ), AMP ( C ) were measured. COC ATP:ADP ( D ), ATP:AMP ( E ) ratios and energy charge ( F ) were calculated. N = 4 biological replicates, data are mean ± SEM. T, treatment; D, oocyte culture duration; *Significantly different (P

    Techniques Used: Cell Culture

    Cellular adenosine metabolism in relation to cAMP-elevating pre-IVM treatment. COC cAMP increases during the peri-ovular period and through pharmacological elevation during pre-IVM. Cyclic AMP is generated by adenylate cyclase (AC) from its substrate ATP and is hydrolysed to AMP by phosphodiesterases (PDE). AMP can be recycled to ATP via the adenosine salvage pathway. The energy sensing enzyme AMP-activated protein kinase (AMPK) is activated by shifts in ATP:AMP and ATP:ADP ratios. CK, creatine kinase; AK, adenylate kinase; Cr, creatine; PCr, phosphocreatine; IBMX, 3-isobutyl-1-methylxanthine; IVM, oocyte in vitro maturation.
    Figure Legend Snippet: Cellular adenosine metabolism in relation to cAMP-elevating pre-IVM treatment. COC cAMP increases during the peri-ovular period and through pharmacological elevation during pre-IVM. Cyclic AMP is generated by adenylate cyclase (AC) from its substrate ATP and is hydrolysed to AMP by phosphodiesterases (PDE). AMP can be recycled to ATP via the adenosine salvage pathway. The energy sensing enzyme AMP-activated protein kinase (AMPK) is activated by shifts in ATP:AMP and ATP:ADP ratios. CK, creatine kinase; AK, adenylate kinase; Cr, creatine; PCr, phosphocreatine; IBMX, 3-isobutyl-1-methylxanthine; IVM, oocyte in vitro maturation.

    Techniques Used: Generated, Polymerase Chain Reaction, In Vitro

    4) Product Images from "The PSMP-CCR2 interactions trigger monocyte/macrophage-dependent colitis"

    Article Title: The PSMP-CCR2 interactions trigger monocyte/macrophage-dependent colitis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05255-7

    PSMP expression was induced by LPS and MDP in CECs, and PSMP promoted the expression of CCL2 in M1 macrophages. ( A – D ) Primary CECs from wild type mice were stimulated with LPS, Pam3CSK4, MDP or ATP for 3 h. PSMP mRNA expression was assessed by real-time PCR. ( E ) During the monocyte-macrophage polarization procession, the PSMP expression in the cell culture medium was detected by CBA and CCL2 expression was detected in parallel by ELISA. ( F ) The CCL2 expression was detected in the medium of M0, M1 and M2 cells with and without PSMP (100 ng/mL) stimulating at the indicated time by ELISA and statistically analyzed. Data were representative of at least three independent experiments. *0.01
    Figure Legend Snippet: PSMP expression was induced by LPS and MDP in CECs, and PSMP promoted the expression of CCL2 in M1 macrophages. ( A – D ) Primary CECs from wild type mice were stimulated with LPS, Pam3CSK4, MDP or ATP for 3 h. PSMP mRNA expression was assessed by real-time PCR. ( E ) During the monocyte-macrophage polarization procession, the PSMP expression in the cell culture medium was detected by CBA and CCL2 expression was detected in parallel by ELISA. ( F ) The CCL2 expression was detected in the medium of M0, M1 and M2 cells with and without PSMP (100 ng/mL) stimulating at the indicated time by ELISA and statistically analyzed. Data were representative of at least three independent experiments. *0.01

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Cell Culture, Crocin Bleaching Assay, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease"

    Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0674-1

    Introduction of mitochondrial dR improves ATP production in dCHCHD2 -deficient flies in a light-dependent manner. a H + -pumping activity of dR WT and dR D104N/K225A (NA) mutant in E. coli . Open and closed triangles represent light on and off, respectively. b Diagram showing light stimulation of mitochondria in flies. Newly eclosed adult files were irradiated with 550 nm LED (7 µW mm − 2 ) at 2 Hz for 12 hr every day. Fly food containing 100 µ m retinal, which were protected from light with aluminum foil, was changed with fresh food every day. c The reduction in ΔΨm in dCHCHD2 −/− neuronal terminals is improved by mito-dR WT but not mito-dR NA. The mitochondria of the abdominal motor neuron terminals of 20-day-old flies visualized with mito-GFP (arrows in upper images, scale bars = 500 µm (left) and 100 µm (right)) were stained with a ΔΨm indicator TMRM (lower images, scale bar = 10 µm). Boxes in the graph indicate the 25th to 75th percentiles, and whiskers represent the maximum and minimum values of the signal intensity of TMRM in the mito-GFP regions. A.U., an arbitrary unit. The numbers of samples analyzed are indicated in the graphs. n = 15–19 flies, Tukey–Kramer test. d Whole-head ATP contents in 15- and 30-day-old male flies. Graphs represent ATP contents normalized against the protein levels. The numbers of samples analyzed are indicated in the graphs. n = 18–22 flies, Tukey–Kramer test. N.S., not significant. e , f Measurement of relative ATP levels (the ratio of 510–580 nm/440–510 nm emission fluorescence intensity) in the cell bodies e and nerve terminals f of PAM cluster DA neurons in 30-day-old males expressing mito-dR WT or NA using the fluorescence ATP biosensor ATeam. All flies were treated with light stimulation. Scale bars = 5 e and 10 f µm. n = 12–13 flies, N.S., not significant by Tukey–Kramer test e . n = 18 flies, two-tailed t test f . Transgenes were driven by D42-GAL4 c , Da-GAL4 d and R58E02-GAL4 e , f . Source data are provided as a Source Data file.
    Figure Legend Snippet: Introduction of mitochondrial dR improves ATP production in dCHCHD2 -deficient flies in a light-dependent manner. a H + -pumping activity of dR WT and dR D104N/K225A (NA) mutant in E. coli . Open and closed triangles represent light on and off, respectively. b Diagram showing light stimulation of mitochondria in flies. Newly eclosed adult files were irradiated with 550 nm LED (7 µW mm − 2 ) at 2 Hz for 12 hr every day. Fly food containing 100 µ m retinal, which were protected from light with aluminum foil, was changed with fresh food every day. c The reduction in ΔΨm in dCHCHD2 −/− neuronal terminals is improved by mito-dR WT but not mito-dR NA. The mitochondria of the abdominal motor neuron terminals of 20-day-old flies visualized with mito-GFP (arrows in upper images, scale bars = 500 µm (left) and 100 µm (right)) were stained with a ΔΨm indicator TMRM (lower images, scale bar = 10 µm). Boxes in the graph indicate the 25th to 75th percentiles, and whiskers represent the maximum and minimum values of the signal intensity of TMRM in the mito-GFP regions. A.U., an arbitrary unit. The numbers of samples analyzed are indicated in the graphs. n = 15–19 flies, Tukey–Kramer test. d Whole-head ATP contents in 15- and 30-day-old male flies. Graphs represent ATP contents normalized against the protein levels. The numbers of samples analyzed are indicated in the graphs. n = 18–22 flies, Tukey–Kramer test. N.S., not significant. e , f Measurement of relative ATP levels (the ratio of 510–580 nm/440–510 nm emission fluorescence intensity) in the cell bodies e and nerve terminals f of PAM cluster DA neurons in 30-day-old males expressing mito-dR WT or NA using the fluorescence ATP biosensor ATeam. All flies were treated with light stimulation. Scale bars = 5 e and 10 f µm. n = 12–13 flies, N.S., not significant by Tukey–Kramer test e . n = 18 flies, two-tailed t test f . Transgenes were driven by D42-GAL4 c , Da-GAL4 d and R58E02-GAL4 e , f . Source data are provided as a Source Data file.

    Techniques Used: Activity Assay, Mutagenesis, Irradiation, Staining, Fluorescence, Expressing, Two Tailed Test

    6) Product Images from "Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization"

    Article Title: Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2019.11.002

    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg His-MBP or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM ATP, were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Figure Legend Snippet: SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg His-MBP or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM ATP, were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.

    Techniques Used: In Vitro, Recombinant, Staining, Immunoprecipitation, Kinase Assay, Incubation

    7) Product Images from "IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation"

    Article Title: IRDL Cloning: A One-Tube, Zero-Background, Easy-to-Use, Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107907

    Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).
    Figure Legend Snippet: Schematic diagram of cloning the gene of interest containing internal restriction sites into expression vector. A, generation of sticky-end fragments and cloning into pWXY1.0 by IRDL cloning. The JcDGAT2 was amplified by two pairs of primers, P1–P2, and P3–P4, which were appended with short sequence tails: C, TCGAG, AATTC, G, respectively. After gel-purification, the two PCR products were mixed together, denatured, and reannealed, resulting in 25% of the DNA fragments with Eco RI and XhoI overhangs. Concomitantly, the vector was double-digested with EcoRI and XhoI for 5–10 min at 37°C. After heat inactivation of the restriction enzyme at 95°C for 5 min, the vector was mixed with the reannealed JcDGAT2 containing EcoRI and XhoI overhangs, T4 DNA ligase and ATP were added and incubated at room temperature for 20 min, and finally transformed into E. coli . B, Restriction digestion (EcoRI and XhoI) of minipreps of pWXY1.0 (V) and pWXY1.0-JcDGAT2 (lane 1 to lane 10). The restriction pattern of pWXY1.0-JcDGAT2 generated by EcoRI and XhoI digestion was as predicted: 861 bp and 200 bp, respectively. M, DNA ruler DL2502 (Generay).

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Amplification, Sequencing, Gel Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Generated

    Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.
    Figure Legend Snippet: Subcloning of one insert into a yeast expression vector pWXY1.0 by using IRDL cloning method. A, Schematic representation of one-step directional cloning of EGFP into a yeast expression vector pWXY1.0. B, Test of the IRDL cloning system. (1): Cloning of EGFP into pWXY1.0 by standard IRDL cloning step (the purified PCR products of GFP and yeast expression vector pWXY1.0 are mixed in a single tube together with FastDigest buffer, restriction enzymes XhoI and KpnI, ATP and T4 DNA ligase, and incubated at 37°C for 30 min), followed by transformation into E. coli Trans 1-T1 as described in materials and methods . (2): A control without T4 DNA ligase. (3): A control without restriction enzymes XhoI and KpnI. C, Colony PCR results from 48 recombinant colonies were run on 1% agarose gels. All of the colonies except the second clone tested contained the correct inserts. M, DNA ruler DL2501 from Generay. D, Plasmids DNA from 23 recombinant colonies and vector pWXY1.0 were digested with XhoI and KpnI and run on 1% agarose gels. DNA from all 23 recombinant colonies displayed the expected restriction pattern of pWXY1.0-EGFP. M, DNA ruler DL2502 (Generay). V, pWXY1.0.

    Techniques Used: Subcloning, Expressing, Plasmid Preparation, Clone Assay, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay, Recombinant

    8) Product Images from "Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay"

    Article Title: Characterization and Optimization of a Red-Shifted Fluorescence Polarization ADP Detection Assay

    Journal: Assay and Drug Development Technologies

    doi: 10.1089/adt.2008.175

    Stability of the FP signal and the ADP detection mixture over time. ( A ) Equilibration of FP signals for ATP/ADP standards representing different percentages of ATP conversions (10 μM ATP/ADP standard curve). Plates were incubated at room temperature
    Figure Legend Snippet: Stability of the FP signal and the ADP detection mixture over time. ( A ) Equilibration of FP signals for ATP/ADP standards representing different percentages of ATP conversions (10 μM ATP/ADP standard curve). Plates were incubated at room temperature

    Techniques Used: Incubation

    ATP/ADP standard curves prepared with Ab1 and Ab2. To mimic ADP generated during an enzyme reaction, standard curves for 0.1 μM , 1 μM , 10 μM , 100 μM , and 1,000 μM ATP were prepared by keeping the adenine concentration
    Figure Legend Snippet: ATP/ADP standard curves prepared with Ab1 and Ab2. To mimic ADP generated during an enzyme reaction, standard curves for 0.1 μM , 1 μM , 10 μM , 100 μM , and 1,000 μM ATP were prepared by keeping the adenine concentration

    Techniques Used: Generated, Concentration Assay

    Impurities in the ATP reagent affected the apparent selectivity of the ADP antibody. ( A ) Competitive binding assays with ADP ( O ) and two vendor sources (V1 [▴] and V2 [▪]) of ATP were performed with Ab1 in ADP Detection Mixture 1. The
    Figure Legend Snippet: Impurities in the ATP reagent affected the apparent selectivity of the ADP antibody. ( A ) Competitive binding assays with ADP ( O ) and two vendor sources (V1 [▴] and V2 [▪]) of ATP were performed with Ab1 in ADP Detection Mixture 1. The

    Techniques Used: Binding Assay

    Control compound screen for assessing assay interference. Test compounds at 10 μM (1% dimethyl sulfoxide) were incubated with 10 m M ATP and a mixture of 1 μM ADP and 9 μM ATP (to represent 10% ATP conversion) in the CK
    Figure Legend Snippet: Control compound screen for assessing assay interference. Test compounds at 10 μM (1% dimethyl sulfoxide) were incubated with 10 m M ATP and a mixture of 1 μM ADP and 9 μM ATP (to represent 10% ATP conversion) in the CK

    Techniques Used: Incubation

    9) Product Images from "Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment"

    Article Title: Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

    Journal: Brain

    doi: 10.1093/brain/awv002

    TPrP induces depletion of intracellular NAD + and ATP. ( A ) TPrP at 5 µg/ml for 3 days induces intracellular NAD + depletion, but not non-toxic PrP at 10 µg/ml. CTRL = untreated cells. Statistical differences are shown compared to CTRL. The
    Figure Legend Snippet: TPrP induces depletion of intracellular NAD + and ATP. ( A ) TPrP at 5 µg/ml for 3 days induces intracellular NAD + depletion, but not non-toxic PrP at 10 µg/ml. CTRL = untreated cells. Statistical differences are shown compared to CTRL. The

    Techniques Used:

    10) Product Images from "The Transcription Bubble of the RNA Polymerase-Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection"

    Article Title: The Transcription Bubble of the RNA Polymerase-Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2012.12.015

    Start-site selection at lacCONS + 2 and lacCONS + 2 derivatives. (a) In vitro transcription reactions using lacCONS + 2 promoters with base-pair substitutions; RNAP, dsDNA, ATP, UTP, CTP and [α 32 P]GTP were incubated in transcription buffer at 37 °C
    Figure Legend Snippet: Start-site selection at lacCONS + 2 and lacCONS + 2 derivatives. (a) In vitro transcription reactions using lacCONS + 2 promoters with base-pair substitutions; RNAP, dsDNA, ATP, UTP, CTP and [α 32 P]GTP were incubated in transcription buffer at 37 °C

    Techniques Used: Selection, In Vitro, Incubation

    11) Product Images from "Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements"

    Article Title: Mitotic chromosomes are constrained by topoisomerase II-sensitive DNA entanglements

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200910085

    Relaxation of mitotic chromosomes by topo II requires hydrolysable ATP. (A) Results of 12 experiments of the type shown in Fig. 1 , showing the ratio of the spring constant after topo II + ATP treatment to native spring constant. More than 20% relaxation occurred in nine of the trials. (B) Results of seven experiments where topo II in AB (no ATP) was sprayed for 30 min. No large relaxations occurred, and in three runs, a slight increase in stiffness was observed. (C) Histogram for 11 experiments where ATP in AB was used for 30 min; in 8/11 experiments, little or no relaxation occurred, but in 3/11 experiments, significant relaxation occurred. (D) Summary of experiments with different enzyme–nucleotide combinations. Height of bars shows relative spring constants normalized to the native (untreated) chromosome spring constant; error bars indicate standard error for each set of experiments. The 35% relaxation effect observed for topo II + ATP (second bar) does not occur for topo II with no ATP (third bar). A small relaxation effect occurs for ATP with no topo II (fourth bar), suggesting that ATPases on the chromosome are active. Experiments with topo II + AMP-PNP (fifth bar) generated nearly no relaxation.
    Figure Legend Snippet: Relaxation of mitotic chromosomes by topo II requires hydrolysable ATP. (A) Results of 12 experiments of the type shown in Fig. 1 , showing the ratio of the spring constant after topo II + ATP treatment to native spring constant. More than 20% relaxation occurred in nine of the trials. (B) Results of seven experiments where topo II in AB (no ATP) was sprayed for 30 min. No large relaxations occurred, and in three runs, a slight increase in stiffness was observed. (C) Histogram for 11 experiments where ATP in AB was used for 30 min; in 8/11 experiments, little or no relaxation occurred, but in 3/11 experiments, significant relaxation occurred. (D) Summary of experiments with different enzyme–nucleotide combinations. Height of bars shows relative spring constants normalized to the native (untreated) chromosome spring constant; error bars indicate standard error for each set of experiments. The 35% relaxation effect observed for topo II + ATP (second bar) does not occur for topo II with no ATP (third bar). A small relaxation effect occurs for ATP with no topo II (fourth bar), suggesting that ATPases on the chromosome are active. Experiments with topo II + AMP-PNP (fifth bar) generated nearly no relaxation.

    Techniques Used: Generated

    Topo II on the chromosomes combined with externally supplied hydrolyzable ATP is responsible for ATP-driven relaxation of trypsinized chromosomes. (A) Force–extension experiment showing initial native elastic response, elasticity after trypsinization, elasticity after subsequent exposure to the topo II inhibitor ICRF-187, and finally exposure to ATP. Data for measurements after each of these chemical exposures are essentially indistinguishable, indicating first that ICRF-187 does not appreciably stiffen or relax chromosomes, and second, that it blocks any relaxation by subsequent exposure to ATP. (B) Results of seven experiments as in A. ATP causes at most a weak relaxation of a trypsinized chromosome after its exposure to ICRF-187 (compare with large effect of ATP in Fig. 4 C ). Bar heights show mean spring constants relative to those of the trypsinized chromosomes. (C) Results of a series of six experiments in which chromosomes were trypsinized, exposed to AMP-PNP, exposed to ATP, and finally exposed to topo II + ATP. Results are normalized to trypsinized chromosomes. AMP-PNP does not change chromosome stiffness (second bar) and blocks relaxation by subsequent ATP exposure (third bar). However, topo II + ATP does relax chromosomes after AMP-PNP exposure (fourth bar).
    Figure Legend Snippet: Topo II on the chromosomes combined with externally supplied hydrolyzable ATP is responsible for ATP-driven relaxation of trypsinized chromosomes. (A) Force–extension experiment showing initial native elastic response, elasticity after trypsinization, elasticity after subsequent exposure to the topo II inhibitor ICRF-187, and finally exposure to ATP. Data for measurements after each of these chemical exposures are essentially indistinguishable, indicating first that ICRF-187 does not appreciably stiffen or relax chromosomes, and second, that it blocks any relaxation by subsequent exposure to ATP. (B) Results of seven experiments as in A. ATP causes at most a weak relaxation of a trypsinized chromosome after its exposure to ICRF-187 (compare with large effect of ATP in Fig. 4 C ). Bar heights show mean spring constants relative to those of the trypsinized chromosomes. (C) Results of a series of six experiments in which chromosomes were trypsinized, exposed to AMP-PNP, exposed to ATP, and finally exposed to topo II + ATP. Results are normalized to trypsinized chromosomes. AMP-PNP does not change chromosome stiffness (second bar) and blocks relaxation by subsequent ATP exposure (third bar). However, topo II + ATP does relax chromosomes after AMP-PNP exposure (fourth bar).

    Techniques Used:

    Related Articles

    Cell Culture:

    Article Title: Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism
    Article Snippet: .. Culture of COCs and denuded oocytes with 13 C5 -AMP In experiments examining conversion of AMP to ATP, COCs or denuded oocytes were cultured for 3 h in bicarbonate-buffered αMEM (Gibco) supplemented with 3 mg/mL BSA, 50 mIU/mL FSH ± ¹³C5 -adenosine 5′-monophosphate (13 C5 -AMP; cat# A281782; Toronto Research Chemicals, Canada) ± 2 mM iodoacetamide (IAC; Sigma) or 200 µM carbenoxolone (CBX; Sigma). .. A concentration of 1 mM ¹³C5 -AMP was used for all experiments except the dose response experiment where doses are indicated.

    Real-time Polymerase Chain Reaction:

    Article Title: The PSMP-CCR2 interactions trigger monocyte/macrophage-dependent colitis
    Article Snippet: .. Primary CECs were plated in 6-well dishes prior to stimulation with LPS (Sigma-Aldrich), Pam3CSK4 (Invivogen), MDP (Invivogen), or ATP (Invitrogen) for 3 h and assessed by real-time PCR. .. The cells in cLP were stained with antibody for flow cytometric analysis.

    Incubation:

    Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease
    Article Snippet: .. TMRM, ATP, and ROS imaging of neurons For tetramethylrhodamine methyl ester (TMRM) staining, the ventral muscles containing the neuromuscular junctions in the abdomen were incubated in 500 nm TMRM (Thermo Fisher Scientific)/HL-3 for 5 min and were washed twice with HL-3. .. TMRM and mito-GFP signals in the abdominal motor neuron terminals were obtained by a laser-scanning microscope system (Leica, TCS-SP5).

    other:

    Article Title: Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes
    Article Snippet: Reagents Fura-4F/acetoxymethyl ester (Fura-4F/AM), pluronic acid, and ionomycin were purchased from Invitrogen; ATP was purchased from Amresco; 2-aminoethyldiphenyl borate (APB) was purchased from Cayman Chemical; vasopressin (Vp) was purchased from Calbiochem; lanthanide (La3+ ) and sulfobromophthalein (BSP) were purchased from Sigma; and thapsigargin (TG) was purchased from Acros Organics.

    Imaging:

    Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease
    Article Snippet: .. TMRM, ATP, and ROS imaging of neurons For tetramethylrhodamine methyl ester (TMRM) staining, the ventral muscles containing the neuromuscular junctions in the abdomen were incubated in 500 nm TMRM (Thermo Fisher Scientific)/HL-3 for 5 min and were washed twice with HL-3. .. TMRM and mito-GFP signals in the abdominal motor neuron terminals were obtained by a laser-scanning microscope system (Leica, TCS-SP5).

    Staining:

    Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease
    Article Snippet: .. TMRM, ATP, and ROS imaging of neurons For tetramethylrhodamine methyl ester (TMRM) staining, the ventral muscles containing the neuromuscular junctions in the abdomen were incubated in 500 nm TMRM (Thermo Fisher Scientific)/HL-3 for 5 min and were washed twice with HL-3. .. TMRM and mito-GFP signals in the abdominal motor neuron terminals were obtained by a laser-scanning microscope system (Leica, TCS-SP5).

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  • 91
    Thermo Fisher cellular atp contents
    The OXPHOS pathway in porcine intestinal epithelial cells is activated by L. gasseri LA39. (A) KEGG pathway enrichment analysis for differentially expressed proteins. Values in the column indicate the normalized p -values (-log10). The most enriched KEGG pathway is displayed at the top of the column. (B) Differential expression profiles of proteins involved in OXPHOS metabolic pathway. All differentially expressed proteins are shown below the corresponding complex in OXPHOS pathway map. Protein marked with a red box showed a significantly increased expression comparing the L. gasseri LA39 group with the Ctrl group. (C) Western blotting analysis of the expression levels of TCIRG1, UQCRC2, and GAPDH in <t>IPEC-J2</t> cells. Normalization and quantitation of TCIRG1/GAPDH and UQCRC2/GAPDH are shown in the corresponding bar chart. (D,E) The relative mRNA expression of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH (D) and UQCRC2/GAPDH (E) were shown in corresponding bar chart. (F) <t>ATP</t> levels of IPEC-J2 cells. The ATP concentration (nmol/L) was normalized to the total protein concentration (mg/L) of WCLs. Data are shown as mean ± SEM; n = 3 (C); n = 5 (E); n = 6 (F). ∗∗ p
    Cellular Atp Contents, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher atp determination intracellular atp levels
    Mitochondrial dysfunction is observed in CD8 + but not <t>CD4</t> + EMRA T cell subsets. (a) Representative flow cytometry plots and cumulative graphs of TMRE staining from middle‐aged donors showing membrane potential in CD45RA/CD27 T cell subsets directly ex vivo defined showing the percentage of cortactin‐positive (a) CD4 + and (b) CD8 + T cells analysed directly ex vivo. Data expressed as mean ± SEM of six donors. (b) Mitochondrial ROS measured using MitoSOX by flow cytometry in CD4 + and CD8 + EMRA T cells from middle‐aged donors. Data expressed as mean ± SEM of six donors. (c) Mitochondrial ROS production expressed as a ratio of mitochondrial mass. Calculated from data shown in Figures 1 b and 2 . (d) γH2AX expression as determined by flow cytometry in CD45RA/CD27‐defined T cell subsets directly ex vivo from middle‐aged donors; the graph shows the mean ± SEM for five donors. (e) Oxygen consumption rates (OCR) of the EMRA CD4 + and CD8 + T cell subsets from middle‐aged donors were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2; the cells were then subjected to a metabolic stress test using the indicated mitochondrial inhibitors. Data are representative of four independent experiments. (f) The basal OCR, extracellular acidification rate (ECAR) and spare respiratory capacity were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2. Graphs show the mean ± SEM for four donors. (g) <t>ATP</t> concentration in EMRA T cell subsets from middle‐aged donors, graphs show the mean ± SEM for five donors. p ‐values were calculated using a t test. * p
    Atp Determination Intracellular Atp Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher pcr atp
    Global assessments of the myocardial bioenergetics of infarcted swine hearts changed in response to combined treatment with a fibrin patch and injected hiPSC-derived cardiac cells. (A) Global assessments of myocardial <t>ATP</t> metabolism were evaluated via an established 31 P MRS-MST experiment. The experiment consists of a control spectrum obtained without saturation ( a1 ) to quantify the magnetization (M0) of phosphocreatine <t>(PCr)</t> and ATPγ, a spectrum obtained with ATPγ saturated ( a2 ) to quantify the steady-state magnetization (Mss) of PCr, and a spectrum obtained with both Pi and PCr saturated ( a3 ) to quantify the steady-state magnetization of ATPγ; saturation was performed with BISTRO frequency-selective pulses applied at the positions indicated by the arrows in spectra a2 and a3 . This approach enables the rates of ATP utilization to be calculated in vivo, even when the resonance for inorganic phosphate (Pi) is obscured by the resonance of 2,3-diphosphoglycerate (2,3-DPG). (B-C) Spectra a1, a2, and a3 were used to calculate global measures of (B) the PCr/ATPγ ratio, and (C) the rate of the PCr→ATP reaction ( k PCR→ATP ) in the hearts of animals in the Cell+Patch, Patch, MI, and Sham groups. * P
    Pcr Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The OXPHOS pathway in porcine intestinal epithelial cells is activated by L. gasseri LA39. (A) KEGG pathway enrichment analysis for differentially expressed proteins. Values in the column indicate the normalized p -values (-log10). The most enriched KEGG pathway is displayed at the top of the column. (B) Differential expression profiles of proteins involved in OXPHOS metabolic pathway. All differentially expressed proteins are shown below the corresponding complex in OXPHOS pathway map. Protein marked with a red box showed a significantly increased expression comparing the L. gasseri LA39 group with the Ctrl group. (C) Western blotting analysis of the expression levels of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH and UQCRC2/GAPDH are shown in the corresponding bar chart. (D,E) The relative mRNA expression of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH (D) and UQCRC2/GAPDH (E) were shown in corresponding bar chart. (F) ATP levels of IPEC-J2 cells. The ATP concentration (nmol/L) was normalized to the total protein concentration (mg/L) of WCLs. Data are shown as mean ± SEM; n = 3 (C); n = 5 (E); n = 6 (F). ∗∗ p

    Journal: Frontiers in Microbiology

    Article Title: Lactobacillus gasseri LA39 Activates the Oxidative Phosphorylation Pathway in Porcine Intestinal Epithelial Cells

    doi: 10.3389/fmicb.2018.03025

    Figure Lengend Snippet: The OXPHOS pathway in porcine intestinal epithelial cells is activated by L. gasseri LA39. (A) KEGG pathway enrichment analysis for differentially expressed proteins. Values in the column indicate the normalized p -values (-log10). The most enriched KEGG pathway is displayed at the top of the column. (B) Differential expression profiles of proteins involved in OXPHOS metabolic pathway. All differentially expressed proteins are shown below the corresponding complex in OXPHOS pathway map. Protein marked with a red box showed a significantly increased expression comparing the L. gasseri LA39 group with the Ctrl group. (C) Western blotting analysis of the expression levels of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH and UQCRC2/GAPDH are shown in the corresponding bar chart. (D,E) The relative mRNA expression of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH (D) and UQCRC2/GAPDH (E) were shown in corresponding bar chart. (F) ATP levels of IPEC-J2 cells. The ATP concentration (nmol/L) was normalized to the total protein concentration (mg/L) of WCLs. Data are shown as mean ± SEM; n = 3 (C); n = 5 (E); n = 6 (F). ∗∗ p

    Article Snippet: Measurement of Cellular ATP Levels The total cellular ATP contents in IPEC-J2 cells and intestinal epithelial cells (including duodenum, jejunum, and ileum) from weaned piglets were measured by an ATP determination kit (Thermo Scientific, A22066) according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Quantitation Assay, Concentration Assay, Protein Concentration

    Fructose increases cellular ATP levels and the release of dopamine from the Neuro 2a cells. The cellular ATP contents (A) , levels of dopamine in culture medium (B) and cell viability (C) of the N2a cells determined at 72 hours after exposure to different concentrations of fructose (0, 12.5, 25, or 50 μM). Values are mean ± SEM of quadruplicate analyses. * P

    Journal: Journal of Biomedical Science

    Article Title: An increase in adenosine-5'-triphosphate (ATP) content in rostral ventrolateral medulla is engaged in the high fructose diet-induced hypertension

    doi: 10.1186/1423-0127-21-8

    Figure Lengend Snippet: Fructose increases cellular ATP levels and the release of dopamine from the Neuro 2a cells. The cellular ATP contents (A) , levels of dopamine in culture medium (B) and cell viability (C) of the N2a cells determined at 72 hours after exposure to different concentrations of fructose (0, 12.5, 25, or 50 μM). Values are mean ± SEM of quadruplicate analyses. * P

    Article Snippet: To determine cellular ATP level in the N2a cells, samples were collected in a lysis buffer (Thermo Fisher Scientific Inc.) and centrifuged at 13500 rpm for 15 min. Supernatant (10 μl) was incubated with ATP reaction mixture for 30 min and detected by the same procedures.

    Techniques:

    KHK gene knockdown prevents the fructose-induced increases in cellular pyruvate and ATP level as well as dopamine release from the N2a cells. Representative immunofluorescence images showing distribution of KHK in the N2a cells (A) on day 7 after the lentiviral transfection of small hairpin RNA of KHK (KHK shRNA) or control shRNA; as well as changes in KHK mRNA (B) , cellular pyruvate (C) and ATP contents (D) , and dopamine levels in the culture medium (E) , detected at 72 hours after the administration of different concentrations of fructose (0, 12.5, 25, or 50 μM) to the culture medium of the KHK shRNA or control shRNA-transfected N2a cells. Values are mean ± SEM of quadruplicate analyses. * P

    Journal: Journal of Biomedical Science

    Article Title: An increase in adenosine-5'-triphosphate (ATP) content in rostral ventrolateral medulla is engaged in the high fructose diet-induced hypertension

    doi: 10.1186/1423-0127-21-8

    Figure Lengend Snippet: KHK gene knockdown prevents the fructose-induced increases in cellular pyruvate and ATP level as well as dopamine release from the N2a cells. Representative immunofluorescence images showing distribution of KHK in the N2a cells (A) on day 7 after the lentiviral transfection of small hairpin RNA of KHK (KHK shRNA) or control shRNA; as well as changes in KHK mRNA (B) , cellular pyruvate (C) and ATP contents (D) , and dopamine levels in the culture medium (E) , detected at 72 hours after the administration of different concentrations of fructose (0, 12.5, 25, or 50 μM) to the culture medium of the KHK shRNA or control shRNA-transfected N2a cells. Values are mean ± SEM of quadruplicate analyses. * P

    Article Snippet: To determine cellular ATP level in the N2a cells, samples were collected in a lysis buffer (Thermo Fisher Scientific Inc.) and centrifuged at 13500 rpm for 15 min. Supernatant (10 μl) was incubated with ATP reaction mixture for 30 min and detected by the same procedures.

    Techniques: Immunofluorescence, Transfection, shRNA

    Mitochondrial dysfunction is observed in CD8 + but not CD4 + EMRA T cell subsets. (a) Representative flow cytometry plots and cumulative graphs of TMRE staining from middle‐aged donors showing membrane potential in CD45RA/CD27 T cell subsets directly ex vivo defined showing the percentage of cortactin‐positive (a) CD4 + and (b) CD8 + T cells analysed directly ex vivo. Data expressed as mean ± SEM of six donors. (b) Mitochondrial ROS measured using MitoSOX by flow cytometry in CD4 + and CD8 + EMRA T cells from middle‐aged donors. Data expressed as mean ± SEM of six donors. (c) Mitochondrial ROS production expressed as a ratio of mitochondrial mass. Calculated from data shown in Figures 1 b and 2 . (d) γH2AX expression as determined by flow cytometry in CD45RA/CD27‐defined T cell subsets directly ex vivo from middle‐aged donors; the graph shows the mean ± SEM for five donors. (e) Oxygen consumption rates (OCR) of the EMRA CD4 + and CD8 + T cell subsets from middle‐aged donors were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2; the cells were then subjected to a metabolic stress test using the indicated mitochondrial inhibitors. Data are representative of four independent experiments. (f) The basal OCR, extracellular acidification rate (ECAR) and spare respiratory capacity were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2. Graphs show the mean ± SEM for four donors. (g) ATP concentration in EMRA T cell subsets from middle‐aged donors, graphs show the mean ± SEM for five donors. p ‐values were calculated using a t test. * p

    Journal: Aging Cell

    Article Title: Mitochondrial mass governs the extent of human T cell senescence. Mitochondrial mass governs the extent of human T cell senescence

    doi: 10.1111/acel.13067

    Figure Lengend Snippet: Mitochondrial dysfunction is observed in CD8 + but not CD4 + EMRA T cell subsets. (a) Representative flow cytometry plots and cumulative graphs of TMRE staining from middle‐aged donors showing membrane potential in CD45RA/CD27 T cell subsets directly ex vivo defined showing the percentage of cortactin‐positive (a) CD4 + and (b) CD8 + T cells analysed directly ex vivo. Data expressed as mean ± SEM of six donors. (b) Mitochondrial ROS measured using MitoSOX by flow cytometry in CD4 + and CD8 + EMRA T cells from middle‐aged donors. Data expressed as mean ± SEM of six donors. (c) Mitochondrial ROS production expressed as a ratio of mitochondrial mass. Calculated from data shown in Figures 1 b and 2 . (d) γH2AX expression as determined by flow cytometry in CD45RA/CD27‐defined T cell subsets directly ex vivo from middle‐aged donors; the graph shows the mean ± SEM for five donors. (e) Oxygen consumption rates (OCR) of the EMRA CD4 + and CD8 + T cell subsets from middle‐aged donors were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2; the cells were then subjected to a metabolic stress test using the indicated mitochondrial inhibitors. Data are representative of four independent experiments. (f) The basal OCR, extracellular acidification rate (ECAR) and spare respiratory capacity were measured following a 15‐min stimulation with 0.5 µg/ml anti‐CD3 and 5 ng/ml IL‐2. Graphs show the mean ± SEM for four donors. (g) ATP concentration in EMRA T cell subsets from middle‐aged donors, graphs show the mean ± SEM for five donors. p ‐values were calculated using a t test. * p

    Article Snippet: 4.7 ATP determination Intracellular ATP levels were measured ex vivo on sorted CD4+ and CD8+ T cell subsets via a bioluminescence assay according to the manufacturer's instructions (Thermo Fisher).

    Techniques: Flow Cytometry, Staining, Ex Vivo, Expressing, Concentration Assay

    Global assessments of the myocardial bioenergetics of infarcted swine hearts changed in response to combined treatment with a fibrin patch and injected hiPSC-derived cardiac cells. (A) Global assessments of myocardial ATP metabolism were evaluated via an established 31 P MRS-MST experiment. The experiment consists of a control spectrum obtained without saturation ( a1 ) to quantify the magnetization (M0) of phosphocreatine (PCr) and ATPγ, a spectrum obtained with ATPγ saturated ( a2 ) to quantify the steady-state magnetization (Mss) of PCr, and a spectrum obtained with both Pi and PCr saturated ( a3 ) to quantify the steady-state magnetization of ATPγ; saturation was performed with BISTRO frequency-selective pulses applied at the positions indicated by the arrows in spectra a2 and a3 . This approach enables the rates of ATP utilization to be calculated in vivo, even when the resonance for inorganic phosphate (Pi) is obscured by the resonance of 2,3-diphosphoglycerate (2,3-DPG). (B-C) Spectra a1, a2, and a3 were used to calculate global measures of (B) the PCr/ATPγ ratio, and (C) the rate of the PCr→ATP reaction ( k PCR→ATP ) in the hearts of animals in the Cell+Patch, Patch, MI, and Sham groups. * P

    Journal: PLoS ONE

    Article Title: 31P NMR 2D Mapping of Creatine Kinase Forward Flux Rate in Hearts with Postinfarction Left Ventricular Remodeling in Response to Cell Therapy

    doi: 10.1371/journal.pone.0162149

    Figure Lengend Snippet: Global assessments of the myocardial bioenergetics of infarcted swine hearts changed in response to combined treatment with a fibrin patch and injected hiPSC-derived cardiac cells. (A) Global assessments of myocardial ATP metabolism were evaluated via an established 31 P MRS-MST experiment. The experiment consists of a control spectrum obtained without saturation ( a1 ) to quantify the magnetization (M0) of phosphocreatine (PCr) and ATPγ, a spectrum obtained with ATPγ saturated ( a2 ) to quantify the steady-state magnetization (Mss) of PCr, and a spectrum obtained with both Pi and PCr saturated ( a3 ) to quantify the steady-state magnetization of ATPγ; saturation was performed with BISTRO frequency-selective pulses applied at the positions indicated by the arrows in spectra a2 and a3 . This approach enables the rates of ATP utilization to be calculated in vivo, even when the resonance for inorganic phosphate (Pi) is obscured by the resonance of 2,3-diphosphoglycerate (2,3-DPG). (B-C) Spectra a1, a2, and a3 were used to calculate global measures of (B) the PCr/ATPγ ratio, and (C) the rate of the PCr→ATP reaction ( k PCR→ATP ) in the hearts of animals in the Cell+Patch, Patch, MI, and Sham groups. * P

    Article Snippet: PCr values obtained from the PCr/ATP of the spectra and chemically determined ATP levels by using a fluorometric ATP Assay Kit (ThermoFisher Scientific, USA) [ ].

    Techniques: Injection, Derivative Assay, Microscale Thermophoresis, Polymerase Chain Reaction, In Vivo

    Spatially localized assessments indicate that the myocardial bioenergetics of swine hearts are disrupted at the border zone of infarction and partially restored by combined treatment with a fibrin patch and injected hiPSC-derived cardiac cells. (A) Spatially localized assessments of myocardial energetics were performed via 31 P 2D-CSI MRS. Spectra were obtained both with (right) and without (left) saturation of the ATPγ resonance and used to calculate (B) the PCr/ATPγ ratios and (C) the rate of the PCr→ATP reaction ( k PCR→ATP ) at the border-zone of infarction and in remote (i.e., noninfarcted) myocardial regions from the hearts of animals in the Cell+Patch and Patch groups. (D) The Flux for the PCr→ATP reaction (Flux PCr→ATP ), as the product of multiplying k PCR→ATP , PCr/ATP ratios, and the ATP level that measured by chemical method. *, P

    Journal: PLoS ONE

    Article Title: 31P NMR 2D Mapping of Creatine Kinase Forward Flux Rate in Hearts with Postinfarction Left Ventricular Remodeling in Response to Cell Therapy

    doi: 10.1371/journal.pone.0162149

    Figure Lengend Snippet: Spatially localized assessments indicate that the myocardial bioenergetics of swine hearts are disrupted at the border zone of infarction and partially restored by combined treatment with a fibrin patch and injected hiPSC-derived cardiac cells. (A) Spatially localized assessments of myocardial energetics were performed via 31 P 2D-CSI MRS. Spectra were obtained both with (right) and without (left) saturation of the ATPγ resonance and used to calculate (B) the PCr/ATPγ ratios and (C) the rate of the PCr→ATP reaction ( k PCR→ATP ) at the border-zone of infarction and in remote (i.e., noninfarcted) myocardial regions from the hearts of animals in the Cell+Patch and Patch groups. (D) The Flux for the PCr→ATP reaction (Flux PCr→ATP ), as the product of multiplying k PCR→ATP , PCr/ATP ratios, and the ATP level that measured by chemical method. *, P

    Article Snippet: PCr values obtained from the PCr/ATP of the spectra and chemically determined ATP levels by using a fluorometric ATP Assay Kit (ThermoFisher Scientific, USA) [ ].

    Techniques: Injection, Derivative Assay, Polymerase Chain Reaction