Detection Of Apoptotic Cells Research Articles

Electronic detection of apoptotic cells on a microchip

Robust and rapid detection of apoptosis in cells is crucially needed for diagnostics, drug discovery, studying pathogenic mechanisms and tracking patient response to medical interventions and treatments. Traditionally, the methods employed to detect apoptosis rely on complex instrumentation like flow cytometers and fluorescence microscopes, which are both expensive and complex-to-operate except in centralized laboratories with trained labor. In this work, we introduce a microfluidic device that can screen cells in a suspension for apoptosis markers and report the assays results as electronic data. Specifically, our device identifies apoptotic cells by detecting externalized phosphatidylserine on a cell membrane – a well-established biomarker that is also targeted by fluorophore-based labeling in conventional assays. In our device, apoptotic cells are discriminated from others through biochemical capture followed by transduction of individual capture events into electrical signals via integrated electrical sensors. The developed technology was tested on simulated samples containing controlled amounts of cells with artificially-induced apoptosis and validated by benchmarking against conventional flow cytometry. Combining sample manipulation and electronic detection on a disposable microfluidic chip, our cell apoptosis assay is amenable to be implemented in a variety of settings and therefore has the potential to create new opportunities for cell-based diagnostics and therapeutics and contribute to improved healthcare outcomes on a large scale.

Involvement of Janus kinase-dependent Bcl-xL overexpression in steroid resistance of group 2 innate lymphoid cells in asthma.

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 μg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s.

Desmoglein-2 Affects Vascular Function in Moyamoya Disease by Interacting with MMP-9 and Influencing PI3K Signaling

The pathogenesis and development of Moyamoya disease are still unclear. This study aimed to investigate the effect of desmoglein-2 (DSG2) on Moyamoya disease and determine the inhibitory effect of DSG2 in vascular remodeling in Moyamoya disease.RNA sequencing, immunohistochemistry (IHC), and western blotting were used to detect the expression of DSG2 in the superficial temporal artery (STA) tissues of Moyamoya disease. The association between DSG2 and endothelial cells’ biological activities was investigated by cell counting kit-8 (CCK-8), migration assay, tube formation assay, flow cytometry with Annexin V-FITC/PI staining, and TUNEL apoptotic cell detection kit. Pathways affected by overexpression or knockdown of DSG2 were identified in endothelial cells.The expression of DSG2 in the STA tissues of Moyamoya disease was lower than that in normal controls. Overexpression of DSG2 inhibits the proliferation and migration but promotes apoptosis in endothelial cells, and low DSG2 levels result in impaired angiogenesis. In addition, there was an interaction between DSG2 and MMP-9, and DSG2 acted through the PI3K signaling in endothelial cells.Our results indicate that DSG2 affects PI3K signaling in vascular endothelial cells, and MMP-9 is involved in DSG2-mediated vascular changes in Moyamoya disease.

Abstract A089: Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells

Abstract Serious concerns include delayed diagnosis and adverse side-effects of conventional treatment leading to residual morbidity in Breast cancer (BC) globally. Traditional Indian medicines/herbs via combination of conventional cancer are gaining increasing acceptance worldwide, playing a pivotal role as proven by their efficacy evaluation studies. Many plants are known to reduce the cell proliferation and tumor development after their treatment. Vardhamana Pippali Rasayana (VPR), an Ayurvedic herbal preparation of pippali (Piper longum) in gradually increasing and tapering dose is used to treat many cancers without any adverse side-effects. However, its anti-cancer role in breast cancer is not known yet. The present study has explored the cytotoxic and apoptotic effects of VPR in triplicates on human breast cancer (MCF7) and normal breast epithelial (MCF10A) cell lines at different concentrations after extracting piperine in aqueous and hydro-alcoholic soxhlet extract (0.25 to 50 μg/μl). The cytotoxic activity was carried out by MTT assay and results showed that a low IC50 was observed at 3.75 μg/μl of VPR against MCF-7 as compared with normal breast epithelial cells. Detection of apoptotic cells using suitable Annexin V was done and there was a time-dependent increase in percentage of annexin V positive cells upon treatment. The above results indicate that VPR may have a strong anti-cancerous potential. But other functional assays are warranted to validate the efficacy of the drug against breast cancer which are under progress in our laboratory. Citation Format: Dr.Richa Tripathi, Shivam Singh, Subhash Gupta, VG Huddar, Tanuja M Nesari, Ashok Sharma. Role of Vardhamana Pippali Rasayana (VPR) in human breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A089.

Apoptosis recognition receptors regulate skin tissue repair in mice.

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1+ macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for efferocytosis pathway genes and display altered efferocytosis signaling via the receptor Axl and its ligand Gas6. During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

Automated detection of apoptotic bodies and cells in label-free time-lapse high-throughput video microscopy using deep convolutional neural networks.

Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent markers of apoptosis, e.g. Annexin-V, that provide an inconsistent and late indication of apoptotic onset for human melanoma cells. Our motivation is to improve the detection of apoptosis by directly detecting apoptotic bodies in a label-free manner. Our trained ResNet50 network identified nanowells containing apoptotic bodies with 92% accuracy and predicted the onset of apoptosis with an error of one frame (5min/frame). Our apoptotic body segmentation yielded an IoU accuracy of 75%, allowing associative identification of apoptotic cells. Our method detected apoptosis events, 70% of which were not detected by Annexin-V staining. Open-source code and sample data provided at https://github.com/kwu14victor/ApoBDproject.

Dual-potential electrochemiluminescence cytosensor based on a metal-organic framework and ABEI-PEI-Au@AgNPs for the simultaneous determination of phosphatidylserine and epidermal growth factor receptors on an apoptotic cell surface.

A new electrochemiluminescence (ECL) cytosensor is proposed for the simultaneous determination of phosphatidylserine (PS) and epidermal growth factor receptor (EGFR) based on the ECL signals of metal-organic framework-5 (MOF-5) loaded CdS quantum dots and N-(aminobutyl)-N-(ethylisoluminol)-polyethylenimine capped Au and Ag nanoparticles. Apoptosis promotes the exposure of PS and reduces the expression of EGFR in cell membranes. Two spatially resolved areas on dual-disk glassy carbon electrodes were designed to eliminate the interference from different ECL probes. Using HepG2 cells treated with resveratrol to induce apoptosis, thecytosensor exhibited high sensitivity, simplicity, and high reproducibility, demonstrating its potential in drug screening and rapid apoptotic cell detection. The strategy reported provides a promising platform for the highly sensitive cytosensing and convenient screening of clinically relevant anticancer drugs.

Hydrogen-rich saline regulates NLRP3 inflammasome activation in sepsis-associated encephalopathy rat model

Sepsis-associated encephalopathy (SAE) is characterised by long-term cognitive impairment and psychiatric illness in sepsis survivors, associated with increased morbidity and mortality. There is a lack of effective therapeutics for SAE. Molecular hydrogen (H2) plays multiple roles in septic diseases by regulating neuroinflammation, reducing oxidative stress parameters, regulating signalling pathways, improving mitochondrial dysfunction, and regulating astrocyte and microglia activation. Here we report the protective effect of hydrogen-rich saline in the juvenile SAE rat model and its possible underlying mechanisms. Rats were injected intraperitoneally with lipopolysaccharide at a dose of 5 mg/kg to induce sepsis; Hydrogen-rich saline (HRS) was administered 1 h after LPS induction at a dose of 5 ml/kg and nigericin at 1 mg/kg 1 h before LPS injection. H&E staining for neuronal damage, TUNEL assay for detection of apoptotic cells, immunofluorescence, ELISA protocol for inflammatory cytokines and 8-OHdG determination and western blot analysis to determine the effect of HRS in LPS-induced septic rats. Rats treated with HRS showed decreased TNF-α and IL-1β expression levels. HRS treatment enhanced the activities of antioxidant enzymes (SOD, CAT and GPX) and decreased MDA and MPO activities. The number of MMP-9 and NLRP3 positive immunoreactivity cells decreased in the HRS-treated group. Subsequently, GFAP, IBA-1 and CD86 immunoreactivity were reduced, and CD206 increased after HRS treatment. 8-OHdG expression was decreased in the HRS-treated rats. Western blot analysis showed decreased NLRP3, ASC, caspase-1, MMP-2/9, TLR4 and Bax protein levels after HRS treatment, while Bcl-2 expression increased after HRS treatment.These data demonstrated that HRS attenuated neuroinflammation, NLRP3 inflammasome activation, neuronal injury, and mitochondrial damage via NLRP3/Caspase-1/TLR4 signalling in the juvenile rat model, making it a potential therapeutic agent in the treatment of paediatric SAE.

Latex C-serum from Hevea brasiliensis induces apoptotic cell death in a leukemic cell line.

Hevea brasiliensis latex is generally cultivated for the use of rubber particles. Previous studies have shown that the antiproliferative activity of C-serum in hepatocellular carcinoma is not induced through the classical apoptotic signaling pathway. However, in a leukemic cell line, the anti-proliferation effect of latex C serum remained unclear. Leukemic cell lines (K562 and U937) and human peripheral blood mononuclear cells (PBMCs) were examined for cell viability using the MTT assay. Flow cytometry was used for apoptotic cell detection by annexin V/PI staining. The expression levels of proapoptotic and antiapoptotic marker genes were measured by qRT‒PCR. Moreover, the caspase activities of the extrinsic and intrinsic apoptotic pathways were detected by enzymatic activities. Latex C-serum inhibited cell proliferation in the K562 and U937 leukemic cell lines but did not affect human PBMCs. Latex C-serum significantly induced the percentage of early and late apoptotic cells in the leukemic cell line. The expression levels of the pro-apoptotic marker genes BAD, BAX, and CASPASE3 significantly increased in the leukemic cell line after post-latex C-serum leukemic cell treatment. The extrinsic, intrinsic and common apoptotic pathways were also studied through caspase-8, -9, and -3 activities. Latex C-serum treatment significantly induced caspase-8, -9, and -3 activation in the K562 cell line and U937 cell line compared to the untreated cells. These results indicate that latex C-serum enhanced anti-proliferation in leukemic cell lines by inducing apoptosis and caspase activation.

Flubendazole exposure disrupts neural development and function of zebrafish embryos (Danio rerio)

Flubendazole (FBZ) is a benzimidazole anthelmintic drug widely used for treating parasitic infections by disrupting microtubule formation and function through tubulin binding. Recently, its use has extended to include anticancer applications, leading to increased environmental exposure to benzimidazole drugs. However, the impact of FBZ on neural development in aquatic organisms, particularly in aquatic vertebrates, remains poorly understood. This study aimed to investigate the potential developmental toxicity of FBZ during neural development using zebrafish model. Various assessments, including analysis of overall developmental changes, morphological abnormalities, apoptosis, gene expression alterations, axon length measurements, and electrophysiological neural function, were performed. FBZ exposure resulted in concentration-dependent effects on survival rate, hatching rate, heartbeat, and the occurrence of developmental abnormalities. Notably, FBZ-induced changes included reductions in body length, head size, and eye size, as well as the detection of apoptotic cells in the central nervous system. Gene expression analysis revealed upregulation of apoptosis-related genes (p53, casp3, and casp8), downregulation of neural differentiation-related genes (shha, nrd, ngn1, and elavl3), and alterations in neural maturation and axon growth-related genes (gap43, mbp, and syn2a). Additionally, shortened motor neuron axon length and impaired electrophysiological neural function were observed. These findings provide novel insights into the potential risks of FBZ on the neural development of zebrafish embryos, emphasizing the need for risk prevention strategies and therapeutic approaches to address the environmental toxicity of benzimidazole anthelmintics.

Potent In Vitro and In Vivo Effects of Stachys lavandulifolia Methanolic Extract against Toxoplasma gondii Infection.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the in vitro effect of the SLME on T. gondii tachyzoites. Totally, 72 male BALB/c mice (40 mice for in vivo evaluation of SLME and 32 mice for its toxicity effects on liver and kidney serum enzymes) were used for the present investigation. At first, 40 mice were orally pre-treated with the SLME at doses of 25, 50, and 75 mg/kg/day for two weeks. Mice were checked daily, and the rate of survival and the mean number of tachyzoites were recorded. Liver lipid peroxidation (LPO) and nitric oxide (NO) levels, the effects on kidney and liver function, as well as the expression level of the proinflammatory cytokines such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ), were studied by the quantitative real-time PCR. Flow cytometry analysis was performed on the effects of SLME on the detection of apoptotic and necrotic cells in T. gondii tachyzoites. The SLME at the concentrations 75 and 150 µg/mL completely killed the tachyzoites after 2 hr of incubation. SLME at 25, 50, and 75 mg/kg/day increased the survival rate of infected mice by the sixth, seventh, and eighth days, respectively. SLME also significantly (p < 0.05) decreased the LPO and NO levels and upregulated the IL-1β and IFN-γ mRNA gene expression levels, whereas no considerable change was observed in the serum level of kidney and liver enzymes. Flow cytometry analysis revealed the prompted early and late apoptosis after exposure to T. gondii tachyzoites with various concentrations of SLME. We found the relevant in vitro anti-Toxoplasma effects of SLME against T. gondii. Moreover, the results confirmed the promising in vivo prophylactic effects of SLME. SLME provokes the innate immune system, induces apoptosis, modulates the proinflammatory cytokines, and inhibits hepatic injury in infected mice. With all these descriptions, further surveys are required to support these findings and elucidate this plant's possible mechanisms of action.

The antitumor effect of mycelia extract of the medicinal macrofungus Inonotus hispidus on HeLa cells via the mitochondrial-mediated pathway.

Inonotus hispidus (I. hispidus), known as shaggy bracket, has been used extensively in China and some East Asian countries as a traditional medicinal macrofungus to treat difficult diseases, such as diabetes, gout, and arthritis. Modern pharmacological research has shown that I. hispidus has an important application value in antitumor treatment. However, the main anti-cervical cancer activity substances from its mycelia and its mechanisms are still not clear. To enrich the germplasm resources of I. hispidus, to reveal the antitumor activity of the extract from the mycelium of I. hispidus against cervical cancer, and to preliminarily analyze its action mechanism. The SH3 strain was isolated from wild fruiting bodies and identified by morphology and molecular biology. The antitumor active component from the mycelium of I. hispidus was isolated and identified with liquid chromatography-tandem mass spectrometry. The cell viability was assessed by MTT assay. The cell cycle distribution, apoptotic cell detection, and mitochondrial membrane potential were detected by flow cytometer. The expression of apoptosis-related proteins was assessed by Western blotting. The inhibition of tumor growth in vivo was assessed by a mouse xenograft model. The SH3 strain was isolated and identified as a new strain of I. hispidus. The antitumor active component containing cyclic peptides from the mycelium of I. hispidus (CCM) was isolated for the first time. In addition, we found that CCM had a strong inhibitory effect on HeLa proliferation in vitro and in vivo. Mechanically, the CCM blocked the cell cycle at the G0/G1 phase, decreased the mitochondrial membrane potential, and eventually promoted apoptosis of HeLa cells through the mitochondria-mediated pathway by upregulating the expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3 and downregulating the expression level of Bcl-2. Our study not only enriches the strain resources of I. hispidus but also confirms that the mycelium of this strain has active components that can inhibit cervical cancer. This is highly significant for the development of active drugs and drug lead molecules for treating cervical cancer.

Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.

Platelet Lysate Therapy Attenuates Hypoxia Induced Apoptosis in Human Uroepithelial SV-HUC-1 Cells through Regulating the Oxidative Stress and Mitochondrial-Mediated Intrinsic Apoptotic Pathway.

(1) Background: Ischemia/hypoxia plays an important role in interstitial cystitis/bladder pain syndrome (IC/BPS). Platelet-rich plasma (PRP) has been shown to relieve symptoms of IC/BPS by regulating new inflammatory processes and promoting tissue repair. However, the mechanism of action of PRP on the IC/BPS bladder remains unclear. We hypothesize that PRP might protect the urothelium during ischemia/hypoxia by decreasing apoptosis. (2) Methods: SV-HUC-1 cells were cultured under hypoxia for 3 h and treated with or without 2% PLTGold® human platelet lysate (PL). Cell viability assays using trypan blue cell counts were examined. Molecules involved in the mitochondrial-mediated intrinsic apoptosis pathway, HIF1α, and PCNA were assessed by Western blot analysis. The detection of apoptotic cells and CM-H2DCFDA, an indicator of reactive oxygen species (ROS) in cells, was analyzed by flow cytometry. (3) Results: After 3 h of hypoxia, the viability of SV-HUC-1 cells and expression of PCNA were significantly decreased, and the expression of ROS, HIF1α, Bax, cytochrome c, caspase 3, and early apoptosis rate were significantly increased, all of which were attenuated by PL treatment. The addition of the antioxidant N-acetyl-L-cysteine (NAC) suppressed the levels of ROS induced by hypoxia, leading to inhibition of late apoptosis. (4) Conclusions: PL treatment could potentially protect the urothelium from apoptosis during ischemia/hypoxia by a mechanism that modulates the expression of HIF1α, the mitochondria-mediated intrinsic apoptotic pathway, and reduces ROS.

Letrozole Decreased Testosterone-Induced Cell Proliferation and Prolactin Secretion also Increased Apoptosis in MMQ and GH3 Rat Prolactinoma Cell Lines.

Aromatase enzyme plays an essential role in estrogen-induced tumorigenesis. It is expressed in the normal pituitary and more significantly in prolactinoma tissues. The aim of this study was to investigate the effects of an aromatase inhibitor, letrozole, on MMQ and GH3 rat prolactinoma cell lines and evaluate the possible mechanism of action. MMQ and GH3 cells were characterized with demonstrating aromatase enzyme and estrogen receptor alpha expression by PCR and immunofluorescence staining. After dose optimization for testosterone (T) and letrozole (L), four groups were established: only the testosteron-treated group (T) to detect cell proliferation; only letrozole-treated group (L) to investigate apoptotic effects; testosterone and letrozole concomitant-treated group to demonstrate inhibition of testosterone induced cell proliferation with letrozole treatment s(T + L) and control group (C) with no treatment. The proliferation rate of cells was determined by WST-1. For the detection of apoptotic and necrotic cells, Annexin V and caspase-3 labeling was used. Prolactin and estrogen levels were measured with ELISA, and the mRNA expression of aromatase and Esr1 was also determined. Testosterone induced the proliferation of MMQ and GH3 cells and further increased prolactin and estradiol levels. Adding letrozole to testosterone resulted in decreased cellular proliferation and even induced apoptosis. Also, letrozole administration significantly decreased prolactin and estradiol levels. However, letrozole alone had no effects on proliferation and apoptosis. Gene expression of aromatase and Esr1 was also significantly decreased by letrozole treatment. This in vitro study demonstrated that treatment of testosterone proliferating cells with letrozole resulted in decreased prolactin levels and cell proliferation and induced apoptosis, and further loss of aromatase and Esr1 mRNA expression were observed. Although this is an in vivo study, the results showed unique and novel findings which may easily be adapted to clinical use for further verification.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Polyvinyl Alcohol can Stabilize FITC Conjugated Recombinant Annexin V for Apoptotic Cells Detection.

Annexin V, a member of calcium-dependent phospholipid-binding proteins, selectively binds to the exposed phosphatidylserine, which can be used for in vitro apoptosis detection. Simultaneous staining of cells with annexin V-fluorescein isothiocyanate (FITC) and the non-vital dye propidium iodide (PI) enables the detection of apoptotic and necrotic cells. Our study aimed to express, purify, and stabilize the recombinant annexin V. The recombinant annexin V was cloned and expressed in E. coli bacteria and was purified using Ni-IDA resin. The FITC conjugation was performed, and apoptosis detection of HaCaT cells by FITC-labeled annexin V was evaluated by flow cytometry. Then, the stability of FITC-labeled annexin in various conditions, including polyvinyl alcohol (PVA), glycerol, and trehalose, was evaluated. The results showed that annexin V was appropriately expressed and purified. After FITC conjugation, it could perfectly detect the cell death of HaCat cells in different apoptosis percentages. FITC-labeled annexin had more stability with PVA than glycerol and trehalose. It seems that PVA has an acceptable effect on FITC-labeled annexin V stability in concentrations lower than 1 mg mL-1 without interfering with fluorescent intensity.

The therapeutic effects of coenzyme Q10 on surgically induced endometriosis in Sprague Dawley rats

The aim of this study was to evaluate the effects of coenzyme Q10 in the treatment of endometriosis rat models. Twenty seven Sprague Dawley rats were divided into four groups; Control Group (n = 7; Endometriosis group), Reference Group (n = 6; Endometriosis + Buserelin acetate, 20 mg/kg), CoQ10 Group-I (n = 7; Endometriosis + CoQ10, 50 mg/kg) and CoQ10 Group-II (n = 7; Endometriosis + CoQ10, 100 mg/kg). At the end of the experiment, all the rats were sacrificed, and the volume and histoarchitecture of endometrial implants were evaluated. The mast cells were determined by Toluidine blue and collagen fiber density was analysed by Masson’s Trichrome staining. Tumour necrosis factor and vascular endothelial growth factor (VEGF) levels were analysed by enzyme-linked immunosorbent assay in peritoneal fluid and VEGF and matrix metalloproteinase-9 (MMP-9) were evaluated by immunohistochemistry. Terminal deoxynucleotidil transferase-mediated dUTP Nick end labelling (TUNEL) was also used for the detection of apoptotic cells. The CoQ10 treatment significantly decreased the volume of endometriotic implants, VEGF, and MMP-9 immunoreactivity and increased TUNEL-positive cells. The findings of the study suggest that CoQ10 can be used in endometriosis treatment by suppressing the endometriotic implants. IMPACT STATEMENT What is already known on this subject? Endometriosis is a gynaecological disorder and previous studies have shown that different treatments with antioxidants cause significant regression in the endometriotic implants. What the results of this study add? In this study, CoQ10 reduced intra-abdominal adhesion scores and volume of the endometriotic implants. In addition, CoQ10 treatment affected mast cell, TNF-α, VEGF, and MMP-9. What of these findings for clinical practice and/or further research? CoQ10 treatments may be possible to apply, it can contribute to science in terms of a new therapeutic treatment for endometriosis. Further studies are required to evaluate the Coenzyme Q10’s effects on pain and subfertility in endometriosis.

Pazopanib restricts small cell lung cancer proliferation via reactive oxygen species-mediated endoplasmic reticulum stress.

BackgroundPazopanib is an approved multitarget anticancer agent for soft tissue sarcoma (STS) and renal cell carcinoma (RCC), which is also under clinical investigation for other malignancies, including small cell lung cancer (SCLC). However, the potential anti‐SCLC mechanisms of pazopanib remain unclear.MethodsCell viability was evaluated by CCK‐8, apoptotic cell detection was conducted using annexin V/PI staining followed by flow cytometry, and Western blot analysis was used to detect the apoptotic‐related molecules and ER‐stress pathway effectors. The intracellular reactive oxygen species (ROS) level was determined by DCFH‐HA staining followed by flow cytometry. An NCI‐H446 xenograft model was established to evaluate pazopanib on tumor suppression in vivo. Immunohistochemistry (IHC) was used to assess the proliferative activity of xenograft in NCI‐H446 cell‐bearing NOD‐SCID mice.ResultsPazopanib dose‐ and time‐dependently inhibited SCLC cell proliferation induced significant apoptosis in SCLC cell lines, increased cleaved‐caspase3 and Bax, and decreased Bcl‐2. Moreover, the PERK‐related ER‐stress pathway was potently activated by pazopanib treatment, inhibiting ER‐stress by salubrinal significantly reversing pazopanib‐mediated apoptosis in SCLC cell lines. Furthermore, pazopanib‐induced intracellular ROS levels increased, while inhibiting ROS by NAC significantly reversed pazopanib‐induced apoptosis in SCLC cells. In addition, pazopanib significantly suppressed NCI‐H446 xenograft growth and decreased Ki67 positive cells in the tumor.ConclusionOur findings indicate that pazopanib induces SCLC cell apoptosis through the ER‐stress process via upregulation of ROS levels. Further investigation of relevant biomarkers to accurately select patients for benefit from pazopanib should be further investigated.

Cell Dissociation Enzymes Affect Annexin V/Flow-Cytometric Apoptotic Assay Outcomes After miRNA-based Transient Transfection.

miRNA functional analysis involves transfection with miRNA-based oligos to identify gain-of or loss-of function cellular phenotypes. Apoptosis is a common phenotypic endpoint for miRNA functional analysis. We report that four common cell dissociation enzymes, TrypLE, Accutase, Trypsin, and Accumax, can differentially impact cell viability and apoptosis in Annexin V flow cytometric analysis after miRNA-based transient transfection. We transiently transfected a nonsense oligo into an epithelial cancer cell line (UM-SCC-12) for 24 h. Cells were harvested with either TrypLE, Accutase, Accumax, or Trypsin after 5 min. The Annexin V/7-AAD assay via flow cytometry was employed. Studies were performed in triplicate. Significant effects were detected by ANOVA, followed by Tukey's Multiple Comparison tests. Trypsin produced the lowest cell viability and lowest percentage of apoptotic cells, specifically when compared to TrypLE and Accutase, respectively (p<0.01). Importantly, transfected trypsinized cells had a significant difference in cell viability and necrosis (p<0.05) when compared with non-transfected trypsinized cells, highlighting the influence of miRNA-based transfection on Annexin V flow cytometric outcomes. Interassay variability was lowest with TrypLE (1.13 %). As such, TrypLE provided the greatest reproducibility and reliability in our cell line. Our study highlights the variable effects of cell dissociation enzymes on transfected cells. Overall, the variability may lead to errors in detection of apoptotic cells using the Annexin V assay after miRNA-based transfection. Before assay use, we recommend pretesting cell dissociation enzymes on transfected cells to ensure reliable and reproducible results.