Seahorse analyzer

OBJECTIVE: To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction (MI) and its correlation with the increase of glycolytic flux. METHODS: Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI. At 28 days after the operation, 5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing...

BACKGROUND: Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence...

Colorectal cancer (CRC) is a prevalent and aggressive malignancy with high mortality rates and significant risks to human well-being. Population-wide screening for tumor suppressor genes and oncogenes shows promise for reducing the incidence and fatality of CRC. Recent studies have suggested that NLRX1, an innate immunity suppressor, may play a role in regulating chronic inflammation and tumorigenesis. However, further investigation is needed to understand the specific role of NLRX1 in CRC. To evaluate the impact of NLRX1 on migration, invasion, and metastasis, two human colon cancer cell lines were studied in vitro...

BACKGROUND: Severe asthma, characterized by inflammation and airway remodeling, involves fibroblast differentiation into myofibroblasts expressing α-SMA. This process leads to the production of fibronectin and connective tissue growth factor (CTGF), driven by factors such as transforming growth factor (TGF)-β. Furthermore, the persistent presence of myofibroblasts is associated with resistance to apoptosis and mitochondrial dysfunction. The chemokine (C-X3-C motif) ligand 1 (CX3CL1) plays a role in tissue fibrosis...

Nitrogen mustard (NM; mechlorethamine) is a cytotoxic vesicant known to cause acute lung injury which can progress to chronic disease. Due to the complex nature of NM injury, it has been difficult to analyze early responses of resident lung cells that initiate inflammation and disease progression. To investigate this, we developed a model of acute NM toxicity using murine precision cut lung slices (PCLS), which contain all resident lung cell populations. PCLS were exposed to NM (1-100 μM) for 0...

BACKGROUND: Ginsenoside Rh2 (G-Rh2), a steroidal compound extracted from roots of ginseng, has been extensively studied in tumor therapy. However, its specific regulatory mechanism in non-small cell lung cancer (NSCLC) is not well understood. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in various malignant tumors. We investigated the impact of G-Rh2 on the malignant progression of NSCLC and how it regulated PDK4 to influence tumor aerobic glycolysis and mitochondrial function...

BACKGROUND: Oral squamous cell carcinoma (OSCC) remains a rampant oral cavity neoplasm with high degree of aggressiveness. Aldo-keto reductase 1B10 (AKR1B10) that is an oxidoreductase dependent on nicotinamide adenine dinucleotide phosphate (NADPH) has been introduced to possess prognostic potential in OSCC. The present work was focused on specifying the involvement of AKR1B10 in the process of OSCC and its latent functional mechanism. METHODS: AKR1B10 expression in OSCC tissues and cells were detected by RT-qPCR and Western blot analysis...

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis...

Prostate cancer is a multi-focal disease that can be treated using surgery, radiation, androgen deprivation, and chemotherapy, depending on its presentation. Standard dose-escalated radiation therapy (RT) in the range of 70-80 Gray (GY) is a standard treatment option for prostate cancer. It could be used at different phases of the disease (e.g., as the only primary treatment when the cancer is confined to the prostate gland, combined with other therapies, or as an adjuvant treatment after surgery). Unfortunately, RT for prostate cancer is associated with gastro-intestinal and genitourinary toxicity...

Atrazine (ATZ), a widely used herbicide with potent endocrine-disrupting properties, has been implicated in hormonal disturbances and fertility issues. Sertoli cells (SCs) play a crucial role in providing mechanical and nutritional support of spermatogenesis. Herein, we aimed to study the effects of environmentally relevant ATZ concentrations on the nutritional support of spermatogenesis provided by SCs. For that, mouse SCs (TM4) were exposed to increasing ATZ concentrations (in μg/L: 0.3, 3, 30, 300, or 3000)...

Adenomyosis is a benign uterine disorder that is associated with female infertility, a reduced clinical pregnancy rate and a high risk of miscarriage. Solute carrier family 38 member a2 (SLC38A2) is a glutamine (Gln) transporter that serves roles in various medical conditions. The present study aimed to reveal the role of SLC38A2 in adenomyosis. The mRNA expression levels of SLC38A2 in eutopic endometrial (EU) and ectopic endometrial (EC) tissues from adenomyotic patients were examined by reverse transcription-quantitative PCR...

Reactive oxygen species (ROS) induces necroptotic and ferroptosis in melanoma cells. Salidroside (SAL) regulates ROS in normal cells and inhibits melanoma cell proliferation. This study used human malignant melanoma cells treated with SAL either alone or in combination with ROS scavenger (NAC) or ferroptosis inducer (Erastin). Through cell viability, wound healing assays, and a Seahorse analyze found that SAL inhibited cell proliferation, migration, extracellular acidification rate, and oxygen consumption rate...

Oxidative stress and mitochondrial dysfunction are important elements for the pathophysiology of preeclampsia (PE), a multisystemic hypertensive syndrome of pregnancy, characterized by endothelial dysfunction and responsible for a large part of maternal and fetal morbidity and mortality worldwide. Researchers have dedicated their efforts to unraveling the intricate ways in which certain molecules influence both energy metabolism and oxidative stress. Exploring established methodologies from existing literature, shows that these investigations predominantly focus on the placenta, identified as a pivotal source that drives the changes observed in the disease...

BACKGROUND: Studies have shown that the chronic use of cannabis is associated with a decrease in blood pressure. Our previous studies prove that activating the cannabinoid type 2 (CB2) receptor in the brain can effectively reduce blood pressure in spontaneously hypertensive rats; however, the exact mechanism has not been clarified. The objective of this study is to demonstrate that activation of microglial CB2 receptors can effectively reduce the levels of TNF-α, IL-1β, and IL-6 in the paraventricular nucleus (PVN) through inhibiting aerobic glycolysis, thereby relieving hypertension...

Efficient metabolism, or the means by which cells produce energy resources, is critical for proper effector function. Here, we present a protocol for examining the bioenergetics and mitochondrial fuel utilization of primary murine autoreactive immunocytes using cellular metabolism-modulating drugs. We describe steps for plate calibration, isolation of primary immunocytes, and Seahorse assay plate preparation. We then detail procedures for performing the XF Cell Mito Stress Test followed by bioenergetics calculations and statistics...

OBJECTIVE: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. METHODS: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates...

Rationale: NPC1 is a protein localized on the lysosome membrane regulating intracellular cholesterol transportation and maintaining normal lysosome function. GWAS studies have found that NPC1 variants in T2D was a pancreatic islet expression quantitative trait locus, suggesting a potential role of NPC1 in T2D islet pathophysiology. Methods: Two-week-old Npc1 -/- mice and wild type littermates were employed to examine pancreatic β cell morphology and functional changes induced by loss of Npc1 . Single cell RNA sequencing was conducted on primary islets...

BACKGROUND: Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed...

BACKGROUND/PURPOSE: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulate), a famous and characteristic Tibetan medicine, has been demonstrated to exert an outstanding brain protection role in the treatment of high-altitude hypoxia disease. However, the metabolic effects of R. crenulate on high-altitude hypoxic brain injury (HHBI) are still incompletely understood. Herein, the anti-hypoxic effect and associated mechanisms of R. crenulate were explored through both in vivo and in vitro experiments...

BACKGROUND: The predominant feature of cancer cells during the process of carcinogenesis is the inclination towards glycolytic metabolism rather than mitochondrial oxidative phosphorylation. Nevertheless, there is a scarcity of research investigating the correlation between bladder cancer and mitochondrial energy metabolism. METHODS: A qPCR array comprising 90 genes associated with mitochondrial oxidative phosphorylation was employed to discern metabolic disparities between three sets of bladder cancer tissue and adjacent normal tissue...