| 1. |
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| 2. |
- Sukhovey, Yurij G., et al.
(författare)
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Difference between the biologic and chronologic age as an individualized indicator for the skin care intensity selection : skin topography and immune system state studies, parameter correlations with age difference
- 2019
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Ingår i: Biomedical Dermatology. - : Springer Nature. - 2398-8460. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Background: Present research addresses the issue of skin aging and corresponding skin treatment individualization. Particular research question was on the developing of simplified criterion supporting patient-specific decision on the necessity and intensity of skin treatment. Basing on the published results and a wide pool of experimental data, we have formulated a hypothesis that a difference between biologic and chronologic age can be used as an express criterion of skin aging.Methods: In present paper, we report the results of studies with 80 volunteers between 15 and 65 years of age, linking parameters reflecting immune state, skin state, and topography to the difference between biologic and chronologic age. Facial skin topography, skin moisture, sebum level, and skin elasticity were studied using commercial devices. Blood immunology studies were performed using venous blood samples. Correlations between all measured parameters and age difference were calculated. Also, cross correlations between skin cell profile and blood immune profile parameters, and skin roughness parameters were calculated.Results: Age dependencies of the blood immunological parameters on the biologic and chronologic age difference are less pronounced as compared to the changes in skin cell profile parameters. However, the changes in the tendencies when biologic age becomes equal to chronologic one are visible for all studied parameters.All measured skin roughness parameters show correlations with age difference, but average skin roughness and depth of the deepest profile valley have the largest correlation coefficient values. Many of the measured skin cell profile and blood immunology parameters show strong correlations with average skin roughness and deepest profile valley, with some of the coefficients exceeding 0.5–0.6.Conclusions: Basing on own experiments and published research results, it is possible to suggest using the difference between calculated biologic age and chronologic age as an individualized criterion supporting decisions on skin treatment strategy. Further research involving larger numbers of participants and aiming on optimizing the expressions for calculating biologic age could lead to reliable and easily available express criterion supporting the decision making for an individualized skin treatment.
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| 3. |
- Lind-Halldén, Christina, et al.
(författare)
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Genetic variation in the syntaxin-binding protein STXBP5 in type 1 von Willebrand disease patients
- 2018
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Ingår i: Thrombosis and haemostasis. - 2567-689X. ; 118:8, s. 1382-1389
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Tidskriftsartikel (refereegranskat)abstract
- von Willebrand factor (VWF) levels in healthy individuals and in patients with type 1 von Willebrand disease (VWD) are influenced by genetic variation in several genes, for example, VWF, ABO and STXBP5. Here, we comprehensively screen for STXBP5 variants and investigate their association with type 1 VWD in Swedish patients and controls. The coding region of the STXBP5 gene was re-sequenced in 107 type 1 VWD patients and the detected variants were genotyped in the type 1 VWD population and a Swedish control population (464 individuals). The functional effects of missense alleles were predicted in silico and the pattern of genetic variation in STXBP5 was analysed. Re-sequencing of 107 type 1 VWD patients identified three missense and three synonymous variants in the coding sequence of STXBP5. The low-frequency missense variants rs144099092 (0.005) and rs148830578 (0.029) were predicted to be damaging, but were not accumulated in patients. No other rare candidate mutations were detected. STXBP5 showed a high level of linkage disequilibrium and a low overall nucleotide diversity of π = 3.2 × 10-4 indicating intolerance to variants affecting protein function. Three previously type 1 VWD-associated single nucleotide polymorphisms were located on one haplotype that showed an increased frequency in patients versus controls. No differences in messenger ribonucleic acid abundance among haplotypes could be found using Genotype-Tissue Expression project data. In conclusion, a haplotype containing the STXBP5 Asn436Ser (rs1039084) mutation is associated with type 1 VWD and no rare STXBP5 mutations contribute to type 1 VWD in the Swedish population.
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| 4. |
- André, Oscar
(författare)
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Data-driven microscopy: placing high-fidelity data in a population-wide context
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mikroskopi är idag ett fundamentalt verktyg inom forskning, där det tillåter oss att skåda in och utforska våra prover i hög detalj. Mycket utav utvecklingen av nya mikroskopimetoder har strävat efter att öka den detaljnivå vi kan uppnå. Samtidigt har utvecklingen inom hårdvara, med tillgång till bättre och mer kraftfulla instrument, lett till utveckligen av metoder där fokuset är att studera en hel population av celler. Till skillnad från när vi studerar ett fåtal celler i hög detalj, tillåter det oss att sätta perspektiv på det vi ser. Det ger oss en förmåga att säga vad det normala beteendet som man kan förvänta sig är, och vilka celler som sticker ut i en population. Med andra ord, vad som är intressant.Samtidigt finns det ett stort intresse av att veta hur varje individuell cell beter sig. Varje cell är, precis som oss människor, unik. De har olika historia, olika ålder och befinner sig i olika tillstånd. Precis som våra celler i kroppen är unika, är även de cellerna som kan orsaka sjukdom unika. För att förstå varför vissa personer är mer känsliga mot sjukdom, och hur en infektion svarar på våra behandlingar behövs en förståelse och an förmåga att studera celler på individuell nivå, samtidigt som vi bibehåller ett perspektiv utifrån populations-nivå.Denna brist på perspektiv har länge varit ett problem inom mikroskopi. Den vanliga lösningen på detta problem är att vi, som människor, kan tolka en bild och peka på vad det är som är intressant eller inte. Vi är, trots allt, extremt duktiga på att tolka visuell information. Men detta är inte en helt felfri lösning. Som människor kan vi vara relativt okonsekventa, vi tolkar oftast utifrån hur vi vill att datan ser ut. Med andra ord, vi saknar förmågan att vara objektiva i vår metodik för att samla in bilder i hög detalj.Min avhandling har till stor del handlat om att utveckla ett verktyg som tillåter oss att sätta perspektiv på det vi studerar med mikroskopi. Detta har lett till Arbete 1, där vi presenterar en allmän strategi (data-styrd mikroskopi) för hur vi kan arbeta med mikroskopi för att samla in data på en hel population, samtidigt som vi kan samla in data med hög detalj på relevanta fynd i populationen. Vi presenterar även här en teknisk lösning, och utför metoden i tre olika scenarion: ett för att studera en population av celler mer allmänt, ett för att fånga det ögonblick som bakterier infekterar mänskliga celler, och ett där vi studerar och fångar in data på relevanta (från ett populations-kontext) cancerceller och följer dem över tid. Denna metod tillåter oss att samla in data i hög detalj på ett objektivt sätt, och att sätta perspektiv på det vi studerar.I Arbete 2 har vi vidare utvecklat på vår metod, där vi försöker lösa problemet att hitta en och samma cell i flera olika mikroskop. Eftersom vi, genom mikroskopi, jobbar på en så ofantligt liten skala, är det oftast väldigt svårt att orientera sig och hitta rätt inom ett prov. Det är lite som att spela På spåret och gissa vart man är, fast utan alla ledtrådar man får på varje nivå. Eftersom vi har tillgång till data på en hel population, så utgick vi från att det borde finnas samband mellan celler och deras grannar i ett prov som är unika för just dem. Genom att använda sig av dessa unika samband kom vi fram med en lösning där vi snabbt kan kalibrera ett prov på ett nytt mikroskop. Det öppnar dörrarna för oss forskare att återanvända prov, att lättare justera provet med nya markörer (för det vi vill visualisera inom cellerna), och att kunna tolka ett prov med data insamlat från flera system.COVID-19 pandemin var en stor omställning för samhället och vården. Likväl var det en stor omställning för många forskningslabb, där en kapplöpning startade för att så snabbt som möjligt förstå sig på hur viruset fungerar och hur vårt immunförsvar svarar på dess infektion. Det var i detta kontext som mitt tredje arbete utfördes. Genom den erfarenhet jag samlat på mig inom mikroskopi och att analysera bilder på stora dataset, bidrog jag med hjälp för att studera hur framtagna antikroppar kan förhindra bindningen av virus-lika partiklar till celler. Antikroppar är ett protein som immunförsvaret producerar i respons mot en patogen. En bättre förståelse kring hur antikroppar verkar, och vad skillnaden mellan en bra och en dålig antikropp är kan leda till framtagningen av bättre vaccin-program och behandlingar inom sjukvården.I Arbete 4 medverkade jag i ett arbete där bakterien Streptococcus pyogenes var i fokus. S. pyogenes enda värd är människor, och ansvarar för över 600 miljoner infektionsfall per år globalt. På bakteriens yta dominerar ett protein, M-proteinet, ett multi-funktionellt protein som bakterien (bland annat) använder sig för att binda till ytor och förhindra immunförsvarets förmåga att göra sig av med bakterien. I arbetet upptäckte vi att fibronektin binder till bakterien (specifikt M-proteinet) olika mycket beroende på mängden antikroppar som finns i miljön. Fibronektin är ett protein som vi människor producerar, och bidrar (bland annat) till att skapa den miljön som celler befinner sig i. Mängden fibronektin varierar beroende på var i kroppen man kollar. Till exempel, i saliv har du en relativt låg mängd fibronektin jämfört med i blodet. Detta ledde till hypotesen att bakterien är special-anpassad för olika miljöer i dess förmåga att undkomma immunförsvaret. En bättre förståelse kring hur bakterien är anpassad till våra olika miljöer och dess infektionsförlopp kan leda till bättre och mer anpassade behandlingar inom sjukvården.
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| 5. |
- Arfvidsson, Berndt, et al.
(författare)
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S100B concentrations increase perioperatively in jugular vein blood despite limited metabolic and inflammatory response to clinically uneventful carotid endarterectomy
- 2015
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Ingår i: Clinical Chemistry and Laboratory Medicine. - : Walter de Gruyter GmbH. - 1434-6621 .- 1437-4331. ; 53:1, s. 111-117
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Tidskriftsartikel (refereegranskat)abstract
- Background: Our aim was to test the hypothesis that metabolic and inflammatory responses of the brain perioperatively during carotid endarterectomy (CEA) might affect blood brain barrier (BBB) integrity.Methods: Twenty patients with >70% stenosis of internal carotid artery (ICA) were prospectively included. Surgery was performed under general anaesthesia. Blood was sampled from ipsilateral internal jugular vein and radial artery: just before, during, and after ICA clamping S100B protein, glucose, lactate, 20 amino acids, and key cytokines were analysed.Results: Jugular vein S100B increased during clamping and reperfusion, while a marginal systemic increase was recorded, unrelated to stump pressure during clamping. Glucose increased during clamping in jugular vein blood and even more systemically, while jugular lactate values were higher than systemic values initially. Most amino acids did not differ significantly between jugular vein and systemic levels: glutamic acid and aspartic acid decreased during surgery while asparagine increased. Jugular vein interleukin (IL)-6 showed a transient non-significant increase during clamping and decreased systemically. IL-8 and IL-10 increased over time.Conclusions: Rising jugular vein S100B concentrations indicated reduced BBB integrity, and marginal secondary increase of S100B systemically. Limited ischaemic effects on the brain during cross-clamping, unrelated to S100B concentrations, were confirmed by lower brain glucose levels and higher lactate levels than in systemic blood. The lack of increased jugular vein glutamic acid disproves any major ischaemic brain injury following CEA. The inflammatory response was limited, did not differ greatly between jugular and systemic blood, and was unrelated to S100B.
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| 6. |
- Cavallaro, Sara, 1992-
(författare)
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Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology.
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| 7. |
- Chantzi, Efthymia, et al.
(författare)
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COMBSecretomics : a pragmatic methodological framework for higher-order drug combination analysis using secretomics
- 2020
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:5
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Tidskriftsartikel (refereegranskat)abstract
- Multi drug treatments are increasingly used in the clinic to combat complex and co-occurring diseases. However, most drug combination discovery efforts today are mainly focused on anticancer therapy and rarely examine the potential of using more than two drugs simultaneously. Moreover, there is currently no reported methodology for performing second- and higher-order drug combination analysis of secretomic patterns, meaning protein concentration profiles released by the cells.Here, we introduce COMBSecretomics (https://github.com/EffieChantzi/COMBSecretomics.git), the first pragmatic methodological framework designed to search exhaustively for second- and higher-order mixtures of candidate treatments that can modify, or even reverse malfunctioning secretomic patterns of human cells. This framework comes with two novel model-free combination analysis methods; a tailor-made generalization of the highest single agent principle and a data mining approach based on top-down hierarchical clustering. Quality control procedures to eliminate outliers and non-parametric statistics to quantify uncertainty in the results obtained are also included. COMBSecretomics is based on a standardized reproducible format and could be employed with any experimental platform that provides the required protein release data. Its practical use and functionality are demonstrated by means of a proof-of-principle pharmacological study related to cartilage degradation.COMBSecretomics is the first methodological framework reported to enable secretome-related second- and higher-order drug combination analysis. It could be used in drug discovery and development projects, clinical practice, as well as basic biological understanding of the largely unexplored changes in cell-cell communication that occurs due to disease and/or associated pharmacological treatment conditions.
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| 8. |
- Chantzi, Efthymia, et al.
(författare)
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Exhaustive in vitro evaluation of the 9-drug cocktail CUSP9 for treatment of glioblastoma using COMBImageDL
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Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514.
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Tidskriftsartikel (refereegranskat)abstract
- The CUSP9 protocol (aprepitant, auranofin, captopril, celecoxib, disulfiram, itraconazole, minocycline, quetiapine, sertraline) is currently undergoing a clinical trial as add-on treatment to standard-of-care temozolomide for recurrent glioblastoma. Although the theoretical repurposing rationale of this 9-drug cocktail is well defined, there is no in vitro experimental data yet supporting its superiority over all its plausible subsets. Such an exhaustive in vitro evaluation may provide preliminary evidence of whether only a fraction of all 9 drugs is needed to achieve an equivalent or even higher effect. Such information could be further used to guide and optimize individualized glioblastoma therapy selection both in terms of efficacy and adverse effects.Here, we employed COMBImageDL, a deep learning improved version of our recently developed COMBImage2 framework, to design, perform and analyze an exhaustive in vitro experiment of the CUSP9 protocol. More specifically, all 511 plausible subsets were evaluated as add-on treatment to temozolomide on a drug resistant glioblastoma cell line (M059K), by combining endpoint cell viability analysis and quantitative live-cell imaging. The experiment was performed in quadruplicate (eight 384-well plates, > 100GB of image data). Fixed clinically achievable concentrations were used for all drugs.Our results suggest that only disulfiram from the CUSP9 cocktail is required, together with temozolomide, in order to induce major changes in cell viability, confluence and morphology. Only slightly increased effects were observed by a few unique higher-order subsets of the CUSP9 protocol, which also contained disulfiram. This finding indicates that for the particular glioblastoma cell line used, the whole CUSP9 protocol could in principle be replaced solely with disulfiram. Notably, it may be worth testing in vitro the few slightly more potent higher-order subsets on primary patient derived glioblastoma cells. This work demonstrates the feasibility and potential of performing exhaustive in vitro evaluation of higher-order drug cocktails prior to subsequent assessment for clinical use. Although the experimental in vitro disease models are not optimal, they can still pinpoint which among all plausible subsets should be further considered. From a personalized therapy selection perspective, in vitro sensitivity testing of primary patient derived tumor cells could thereby advance from the current practice based on single drugs and only cytotoxicity readouts to also include higher-order drug cocktails and quantitative live-cell imaging.
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| 9. |
- Cholujová, Dana, et al.
(författare)
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Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays
- 2008
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Ingår i: Immunobiology. - : Elsevier. - 0171-2985 .- 1878-3279. ; 213:8, s. 629-640
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Tidskriftsartikel (refereegranskat)abstract
- Cytotoxicity is one of the major defence mechanisms against both virus-infected and tumor cells. Radioactive 51chromium (51Cr) release assay is a “gold standard” for assessment of natural killer (NK) cytolytic activity in vitro. Several disadvantages of this assay led us to design alternative tools based on flow cytometry analysis. Four different fluorescent dyes, calcein acetoxymethyl ester (CAM), carboxyfluorescein succinimidyl ester (CFSE), Vybrant DiO (DiO) and MitoTracker Green (MTG) were tested for labeling of NK target K-562 cells. Target staining stability, spontaneous release of fluorochromes and subsequent accumulation in bystander unstained cells were measured using fluorimetry and flow cytometry. Healthy donor peripheral blood mononuclear cells and affinity column purified NK cells were used as effectors coincubated with target K-562 cells at different E:T ratios for 3h and 90min, respectively. Fluorescent probe 7-amino-actinomycin D was used for live and dead cell discrimination. Bland–Altman statistical method was applied to measure true agreement for all CAM–51Cr, CFSE–51Cr, DiO–51Cr and MTG–51Cr pairs analyzed. Based on the data, none of the four proposed methods can be stated equivalent to the standard 51Cr release assay. Considering linear relationships between data obtained with four fluorochromes and 51Cr release assay as well as linear regression analysis with R2=0.9393 value for CAM–51Cr pair, we found the CAM assay to be the most closely related to the 51Cr assay.
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| 10. |
- Christoffersson, Jonas, 1986-, et al.
(författare)
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A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device
- 2018
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Ingår i: Bioengineering. - : MDPI AG. - 2306-5354. ; 5:2, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Three-dimensional (3D) models with cells arranged in clusters or spheroids have emerged as valuable tools to improve physiological relevance in drug screening. One of the challenges with cells cultured in 3D, especially for high-throughput applications, is to quickly and non-invasively assess the cellular state in vitro. In this article, we show that the number of cells growing out from human induced pluripotent stem cell (hiPSC)-derived cardiac spheroids can be quantified to serve as an indicator of a drug’s effect on spheroids captured in a microfluidic device. Combining this spheroid-on-a-chip with confocal high content imaging reveals easily accessible, quantitative outgrowth data. We found that effects on outgrowing cell numbers correlate to the concentrations of relevant pharmacological compounds and could thus serve as a practical readout to monitor drug effects. Here, we demonstrate the potential of this semi-high-throughput “cardiac cell outgrowth assay” with six compounds at three concentrations applied to spheroids for 48 h. The image-based readout complements end-point assays or may be used as a non-invasive assay for quality control during long-term culture.
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