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PubMed1992

Early midzonal oxidative stress preceding cell death in hypoperfused rat liver.

Intralobular heterogeneity of oxidative stress and its topographic relationship with cell death during low-flow hypoxia were shown in perfused rat liver by digital microfluorography using dichlorofluorescin diacetate, a fluorochrome sensitive to intracellular hydroperoxide formation, and propidium iodide, which labels the nuclei of nonviable cells. The surface of the liver loaded with two precursors was microscopically visualized, and the fluorescence of dichlorofluorescein, a highly fluorescent molecule generated by hydroperoxide-mediated dichlorofluorescin oxidation, was digitally processed. Dichlorofluorescein fluorescence significantly increased in midzonal regions as early as 20 minutes after starting the 25% low-flow hypoxia. At 40 minutes the fluorograph showed multiple dotted patterns, and the intensity peaked at 60 minutes. The onset of cell death studied by propidium iodide was observed at 40 minutes, and its topographic distribution corresponded to the dichlorofluorescein-enhanced midzonal regions. Allopurinol diminished the early midzonal oxidative stress and retarded the onset of cell death. The current findings show that xanthine oxidase-dependent oxidative stress and the resultant cell death during low-flow hypoxia are spatially restricted in the intermediate zone between the periportal and pericentral regions.

Animals↗
PubMed1988

Limitations of flow cytometry for the specific detection of bacteria in mixed populations.

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.

Antibodies, Bacterial↗
PubMed1990

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells.

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells. F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37 degrees C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37 degrees C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained. A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.

Cell Line↗
PubMed1995

A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity.

A flow cytometry based method has been developed to assess natural killer (NK) cell activity in both short-term (4 h) and long-term (18 h) NK assays. Target cells were either labeled with PKH-2, c'FDA or D275. Simultaneously, dead cells were identified by counter-staining with the nuclear dye propidium iodide. Using flow cytometry, only D275 in combination with propidium iodide permits the differentiation of four cell populations: live target cells, dead target cells, live effector cells, and dead effector cells. Even after the extended incubation periods (18 h) necessary for the determination of NK activity in some domestic animals these four populations remain clearly distinguishable. Comparison of results with cells of normal human individuals obtained using this D275/propidium iodide flow cytometry assay with data derived from fluorescence microscopy or an endogenous lactate dehydrogenase release assay shows a strong correlation. Since in long-term NK assays a high proportion of dead effector cells is constantly observed this cell population frequently limits the use of the lactate dehydrogenase release assay but does not interfere with the flow cytometry assay presented here. Using this novel assay, we have demonstrated the suppressive effects of defined glycosaminoglycans on long-term porcine NK activity.

Animals↗
PubMed1995

A simplified method for the coordinate examination of apoptosis and surface phenotype of murine lymphocytes.

Murine lymphocytes readily undergo spontaneous and glucocortocoid-induced apoptosis in vitro. It has been previously demonstrated that during apoptosis, many cell types including lymphocytes, enzymatically cleave their DNA, thus demonstrating a sub-G0 DNA peak when stained with propidium iodide and analyzed by flow cytometry. In a mixed population, it is often desirable to phenotypically identify distinct populations or subsets undergoing apoptosis, thus requiring multiparameter analysis of surface phenotype and DNA content. Paraformaldehyde fixation procedures, although common for surface evaluation, have not been extensively used in methods quantifying apoptosis. To measure apoptosis in a mixed lymphocyte population, we evaluated a gentle detergent permeabilization and paraformaldehyde fixation procedure combined with propidium iodide (PI) DNA staining, adapted from existing methods for cell cycle studies. With this method and rigorous gating techniques which we defined, we detected both apoptotic and debris fractions within the sub-G0 cell cycle region of a glucocortocoid-treated murine lymphocyte cell line. Using this cell line, WEHI 231.7, as a lymphocyte model, we developed a logical gating strategy to exclude debris from analysis. We further demonstrated that apoptosis in freshly isolated murine lymphocytes detected with paraformaldehyde fixation and PI staining was quantitatively comparable to PI staining with ethanol fixation, or nick translation labeling of DNA strand breaks (TUNEL). Finally, using fresh murine spleen cells, we demonstrated that paraformaldehyde fixation preserves surface protein staining, allowing multiparameter analysis of immunophenotype and apoptotic or cell cycle status in a mixed lymphocyte population. Thus, this method offers an inexpensive and technically simple alternative for assessing apoptosis and surface phenotype.

Animals↗
PubMed1995

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance.

Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.

Acetylcholinesterase↗
PubMed2006

New multipotent tetracyclic tacrines with neuroprotective activity.

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5-13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca(2+) overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of beta-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70mM K(+) and 60microM H(2)O(2). Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

Acetylcholinesterase↗
PubMed2006

DRAQ5: improved flow cytometric DNA content analysis and minimal residual disease detection in childhood malignancies.

BACKGROUND: The majority of flow cytometric DNA content analyses are performed on whole peripheral blood, bone marrow or tumor samples containing significant numbers of non-malignant cells which hamper specific DNA content analysis. Simultaneous analysis of DNA content and immunophenotype greatly improves the specificity of DNA-ploidy measurements. Therefore, a three-color flow cytometric assay using DRAQ5 was developed and validated. METHODS: The results of DNA content analysis using DRAQ5 and propidium iodide were compared using peripheral blood samples of 15 healthy volunteers. The reproducibility of the multiparameter DRAQ5 assay was assayed in 10 analytical runs and the sensitivity was evaluated with addition experiments. RESULT: Using DRAQ5, slightly wider CVs of the G0/G1 peak were obtained (average CV=3.29%). When DRAQ5 staining and immunophenotyping were combined, the within- and between-run imprecision was 1.98% and 1.67%, respectively and the DNA content of 25 DNA-hyperdiploid tumor cells (DNA-index=1.16) per 4.10(4) mononuclear cells (0.06%) could be determined. CONCLUSIONS: The multiparameter DRAQ5 assay has a superior sensitivity and specificity compared to the propidium iodide based method. Since at least 25 DNA-hyperdiploid cells per 10(4) DNA-diploid mononuclear cells could be detected, multiparameter DRAQ5 DNA content analysis could be used to study minimal residual disease.

Anthraquinones↗
PubMed2004

The application of image cytometry to viability assessment in dual fluorescence-stained fish spermatozoa.

The viability of spermatozoa has been assessed using SYBR 14 staining for DNA of living cells and propidium iodide staining for DNA of degenerate cells. This dual staining was performed on four fish species (Siberian sturgeon, Acipenser baerii; common carp, Cyprinus carpio; tench, Tinca tinca and wels, Silurus glanis) and the proportions of live and dead spermatozoa were assessed by epifluorescence microscopy and image cytometry. Ten phase contrast and epifluorescent images were recorded per sample, corresponding images were overlaid, and the blended images were evaluated for live and dead spermatozoa, represented by green and red fluorescence signals. Live/dead proportions were assessed, after dual thresholding, by imaging software that counted absolute numbers of objects and computed their frequencies. All sperm heads were found to be labelled, emitting either green or red light. Mean numbers of spermatozoa per image were in the ranges 32-113, 61-105, 48-104 and 29-91 for Siberian sturgeon, common carp, tench and wels, respectively. The corresponding proportions of live spermatozoa were in the ranges 83.56-94.59%, 93.92-97.02%, 76.14-97.76% and 79.45-83.76%. Standard deviations did not exceed 5% of the means. The image cytometric system using dual staining with SYBR 14 and propidium iodide was clearly suitable for assessing the viability of freshwater fish spermatozoa.

Animals↗
PubMed2004

Sperm mitochondrial DNA depletion in men with asthenospermia.

OBJECTIVE: To determine the content of sperm mitochondrial DNA (mtDNA) in patients with asthenospermia or with poor sperm motility. DESIGN: Analysis of the content of mtDNA as the ratio between the amount of mtDNA and nuclear DNA by using a new concurrent polymerase chain reaction. SETTING: University hospital infertility center. PATIENT(S): Eighty-six men who sought infertility therapy. INTERVENTION(S): Moving characteristics of sperm were examined with a computer-assisted semen analyzer. MAIN OUTCOME MEASURE(S): Sperm samples were classified into two groups, one with asthenospermia and the other with normal moving characteristics. The content of mtDNA in sperm was determined by polymerase chain reaction. We analyzed the mitochondrial mass by MitoTracker Green staining and analyzed DNA content with propidium iodide staining by flow cytometry. RESULT(S): A decrease in sperm mtDNA content was detected in patients with asthenospermia or with poor sperm motility (<20% motility). The relative mtDNA contents in the asthenospermia and normal groups were 7.2 +/- 1.3 (mean +/- SD, n = 23) and 74.1 +/- 2.0 (n = 29), respectively. Lower intensities of propidium iodide staining were detected in patients with asthenospermia or poor motility, but there was no significant difference in MitoTracker Green staining between the sperm with different motility. CONCLUSION(S): We suggest that mtDNA content may serve as a useful indicator of sperm quality and that mtDNA depletion may play an important role in the pathophysiology of some male infertility.

Case-Control Studies↗
PubMed2006

Microvascular permeabilization and cardiomyocyte injury provoked by myocardial contrast echocardiography in a canine model.

OBJECTIVES: The aim of this research was to evaluate the potential for myocardial contrast echocardiography (MCE) to provoke microscale bioeffects in a canine model. BACKGROUND: Myocardial contrast echocardiography induces bioeffects in rat hearts, but translation of such results to larger animal models is uncertain. METHODS: Dogs were anesthetized and prepared for open- (n = 22) or closed- (n = 6) chest MCE. Evans blue dye was injected intravenously as an indicator of microvascular leakage, and propidium iodide was used to stain for irreversibly injured myocytes in frozen sections. The contrast agent (Definity, Bristol-Myers Squibb Medical Imaging Inc., North Billerica, Massachusetts) was diluted in saline and infused intravenously at 2 microl/kg/min. Myocardial contrast echocardiography in a short-axis (open-chest) or modified four-chamber view (closed-chest) with 1:4 end systolic electrocardiogram triggering was performed at 1.5 MHz for 10 min in a single imaging plane. RESULTS: Petechiae and leakage of Evans blue were observed in the ultrasound scan plane within the anterior left ventricle. For 1.2 MPa and 2.2 MPa, open- or closed-chest MCE, Evans blue content in tissue within the scan plane was significantly greater than in tissue outside this plane. Counts of propidium-iodide-stained nuclei for 2.2 MPa open-chest MCE were also significantly greater inside than outside the scan plane. CONCLUSIONS: In a canine model, MCE induces myocardial injury comparable to that seen in the rodent model.

Animals↗
PubMed2006

A rapid method to measure beta-amyloid induced neurotoxicity in vitro.

Beta-amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to be associated with neurotoxicity in the disease. Abeta-induced neurotoxicity is strongly dependent on its structure. The aggregation of the peptide from monomeric form to fibrils is a function of many variables including time. It is because of this dynamic nature of Abeta structure that there is a necessity for an in vitro toxicity assay that is rapid enough to eliminate or at least lower the possibility of Abeta structural changes during the time required for the assay. Here we describe a fast and sensitive method with which to assess Abeta-induced neurotoxicity in SH-SY5Y neuroblastoma cells. The method employs two-color flow cytometry using annexin-phycoerythrin, a marker for cell surface phosphatidylserine that typically indicates early stages of apoptosis, and 7-amino-actinomycin D, a membrane impermeant nucleic acid dye. We compare results using the two-color assay to those obtained using the propidium iodide toxicity assay and demonstrate comparability of results, but in 2h or less with the two-color assay as opposed to 24-48 h with the propidium iodide assay. The assay described could be a useful tool in evaluating the role of Abeta structure in biological activity of the peptide.

Amyloid beta-Peptides↗
PubMedDate not supplied

Ischemic tolerance induction in organotypic hippocampal slices: role for the GABA(A) receptor?

Ischemic preconditioning (IPC) refers to sublethal ischemic insults rendering brain tissue tolerant against subsequent ischemic insults. We investigated the role of the GABA(A) receptor (GABA(A)R) upon IPC induction. Rat organotypic hippocampal slices were subjected to IPC by 15 min of oxygen-glucose deprivation (OGD) followed by 40 min of OGD 48 h later, resulting in robust cell death reduction as assessed by the propidium iodide fluorescence method ('late' or 'second window' IPC). Superfusion with the GABA(A)R antagonist bicuculline during IPC ameliorated propidium iodide uptake at a high but not at low doses indicating that GABA(A)R activation may be assigned a limited role in neuroprotection. In previous studies, we found that increased neuronal excitability can promote IPC neuroprotection. We, therefore, tested the hypothesis that blockade of inhibitory GABAergic transmission conferred ischemic tolerance. However, temporary administration of bicuculline 48 h prior to ischemic challenge was not neuroprotective. In another approach, we tested whether preconditioning with the GABA(A)R agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) mediated ischemic tolerance and found no significant neuroprotection. The results are discussed in light of the intrinsic excitatory-inhibitory balance of glutamate and GABA.

Animals↗
PubMed2006

Estrogen preconditioning protects the hippocampal CA1 against ischemia.

Estrogen is neuroprotective against ischemia in both in vivo and in vitro injury models. Because of the promising preclinical data on neuroprotection, the Women's Estrogen for Stroke Trial was initiated. The outcomes from this trial were, however, unsuccessful and questions emerged about the safety of chronic estrogen treatment in women. In contrast to the chronic estrogen treatment strategy, the present study aims to investigate: (1) the neuroprotective efficacy of single estrogen pretreatment/preconditioning; and (2) the existence of a similarity between estrogen- and ischemic preconditioning-induced neuroprotection against cerebral ischemia. The efficacy of estrogen was tested in an in vitro model of cerebral ischemia using hippocampal organotypic slice culture system. The hippocampal organotypic slice cultures were generated from female neonatal (9-11 days old) Sprague-Dawley rats. The slices were exposed to estradiol-17beta (0.5, 1, 5 nM) for various durations (1, 2 or 4 h) 48 h prior to ischemia (40 min of oxygen-glucose deprivation). For ischemic preconditioning, slices were exposed to sublethal oxygen-glucose deprivation (15 min), 48 h prior to lethal oxygen-glucose deprivation. Quantification of cell death in hippocampal CA1 region was conducted by using propidium iodide fluorescence staining technique. Results demonstrated that estrogen preconditioning significantly protects the hippocampal CA1 region against ischemia (P<0.001) and mimicked ischemic preconditioning-induced neuroprotection. The propidium iodide fluorescence values of estrogen preconditioning, ischemic preconditioning and ischemia groups were 21+/-2 (mean+/-S.E.M.) (1 nM; 2 h; n=15), 18+/-2 (5 nM; 4 h; n=12), 32+/-3 (n=8), 65+/-3 (n=27), respectively. Further, estrogen preconditioning initiated a calcium-mediated signaling pathway leading to protection of CA1 neurons against ischemia. Future investigations in estrogen preconditioning may suggest new estrogen regimens that avoid potential side effects of chronic estrogen treatment for stroke patients.

Analysis of Variance↗
PubMed2006

Allicin, a major component of garlic, inhibits apoptosis of macrophage in a depleted nutritional state.

OBJECTIVE: Allicin is believed to be the main component responsible for the biological activity of garlic. The regulation of cell death might have therapeutic potential in many diseases, and previous studies have demonstrated that allicin stimulates the functional activity of macrophages. Therefore, this study examined the effects of allicin on the apoptosis of macrophages induced by serum- and amino acid-free culture. METHODS: The apoptosis of peritoneal macrophages was examined after pretreating them with allicin and incubating them in a depleted nutritional state. The rate of apoptosis was determined using propidium iodide staining analysis using flow cytometry, DNA fragmentation, and a caspase-3 assay. Western blot analysis was used to examine the changes in the pro- or antiapoptotic protein expression levels. RESULTS: DNA fragmentation and propidium iodide staining analyses revealed that allicin decreased the malnutrition-induced apoptosis of macrophages. The level of Bax expression, the amount of cytochrome-c released from the mitochondria, and the caspase-3 activity were also lower in the allicin-treated macrophages than in the untreated macrophages in a depleted nutritional state. Moreover, the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase) inhibitor suppressed the allicin-induced inhibition of apoptosis in a depleted nutritional state and allicin increased the level of ERK1/2 phosphorylation. CONCLUSION: Allicin inhibits the apoptosis of macrophages in a depleted nutritional state through the MEK/extracellular signal-regulated kinase pathway.

Animals↗
PubMed2004

Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development.

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.

Animals↗
PubMed2005

Increase in intracellular Cd(2+) concentration of rat cerebellar granule neurons incubated with cadmium chloride: cadmium cytotoxicity under external Ca(2+)-free condition.

In order to examine the cadmium cytotoxicity unrelated to external Ca(2+), the effects of micromolar CdCl(2) on intracellular Cd(2+) concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca(2+) and external Ca(2+)-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl(2) (10-300 microM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl(2) equally enhanced the fluo-3 fluorescence under both Ca(2+) conditions and MnCl(2) did not quench the CdCl(2)-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd(2+) concentration. CdCl(2) at 100-300 microM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100-300 microM CdCl(2). Similar results described above were also observed under external Ca(2+)-free condition. It is suggested that some of cytotoxic actions of CdCl(2) on neurons are unrelated to external Ca(2+), one of main sources for increasing intracellular Ca(2+) concentration.

Aniline Compounds↗
PubMed2003

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Adenosine Triphosphate↗