Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress

Bondza-Kibangou, Patrick; Millot, Christine; Dufer, Jean; Millot, Jean-Marc

doi:10.1016/s0248-4900(01)01135-2

Cited by 13 publications

(10 citation statements)

References 30 publications

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“…[23-25]. The exact pattern and level of AF is related to the cell type, functional state of the cell, cellular morphological features, and fixation status of the specimen [26,27]. Because of its broad spectrum, AF can be distinguished from specific fluorescence emitted by fluorochromes and/or FPs.…”

Section: Discussionmentioning

confidence: 99%

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Vorobjev

Buchholz

Prabhat³

et al. 2012

Malar J

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BackgroundMalaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP.MethodsDetection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry.ResultsA new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites.DiscussionFilter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events.

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Section: Discussionmentioning

confidence: 99%

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Vorobjev

Buchholz

Prabhat³

et al. 2012

Malar J

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“…Autofluorescence (esp. with emission between 450 and 550 nm) has been observed to vary in "stressed" mitochondria (e.g., oxidative stress, radiation stress, protein aggregation) compared to unstressed controls (Andersson, Baechi, Hoechl, & Richter, 1998;Bartolomé and Abramov, 2015;Bondza-Kibangou, Millot, Dufer, & Millot, 2001;Kuznetsov, Margreiter, Amberger, Saks, & Grimm, 2011;Layfield et al, 2006;Monici, 2005;Schaue, Ratikan, & Iwamoto, 2012). Changes in fluorescence have been attributed to altered NADH/NAD(P)H ratio and the presence of oxidized FADH 2 , which during mitochondrial ATP production, serve as electron carriers to complexes I and II, respectively.…”

Section: Troubleshootingmentioning

confidence: 99%

Mitochondrial Subtype Identification and Characterization

2018

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Healthy, functional mitochondria are central to many cellular and physiological phenomena, including aging, metabolism, and stress resistance. A key feature of healthy mitochondria is a high membrane potential (Δψ) or charge differential (i.e., proton gradient) between the matrix and inner mitochondrial membrane. Mitochondrial Δψ has been extensively characterized via flow cytometry of intact cells, which measures the average membrane potential within a cell. However, the characteristics of individual mitochondria differ dramatically even within a single cell, and thus interrogation of mitochondrial features at the organelle level is necessary to better understand and accurately measure heterogeneity. Here we describe a new flow cytometric methodology that enables the quantification and classification of mitochondrial subtypes (via their Δψ, size, and substructure) using the small animal model C. elegans. Future application of this methodology should allow research to discern the bioenergetic and mitochondrial component in a number of human disease and aging models, including, C. elegans, cultured cells, small animal models, and human biopsy samples. © 2018 by John Wiley & Sons, Inc.

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“…The autofluorescence emission of some of these, most notably NAD(P)H, depends on its oxidation state. NAD(P)H is fluorescent wheras NAD+ is not, and the relative balance of these two depends on the level of ROS [18,19]. This allows the status of cellular, mitochondrial or cytosolic ROS to be non-invasively evaluated and monitored [20].…”

Section: Introductionmentioning

confidence: 99%

Non-invasive real-time imaging of reactive oxygen species (ROS) using multispectral auto-fluorescence imaging technique: a novel tool for redox biology

Habibalahi¹,

Moghari

Campbell

et al. 2020

Preprint

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AbstractDetecting reactive oxygen species (ROS) that play a critical role as redox modulators and signalling molecules in biological systems currently requires invasive methods such as ROS - specific indicators for imaging and quantification. We developed a non-invasive, real-time, label-free imaging technique for assessing the level of ROS in live cells and thawed cryopreserved tissues that is compatible with in-vivo imaging. The technique is based on autofluorescence multispectral imaging (AFMI) carried out in an adapted fluorescence microscope with an expanded number of spectral channels spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. We established a strong quantitative correlation between the spectral information obtained from AFMI and the level of ROS obtained from CellROX staining. The results were obtained in several cell types (HeLa, PANC1 and mesenchymal stem cells) and in live kidney tissue. Additioanly, two spectral regimes were considered: with and without UV excitation (wavelengths > 400 nm); the latter being suitable for UV-sensitive systems such as the eye. Data were analyzed by linear regression combined with an optimization method of swarm intelligence. This allowed the calibration of AFMI signals to the level of ROS with excellent correlation (R= 0.84, p=0.00) in the entire spectral range and very good correlation (R= 0.78, p=0.00) in the limited, UV-free spectral range. We also developed a strong classifier which allowed us to distinguish moderate and high levels of ROS in these two regimes (AUC= 0.91 in the entire spectral range and AUC = 0.78 for UV-free imaging). These results indicate that ROS in cells and tissues can be imaged non-invasively, which opens the way to future clinical applications in conditions where reactive oxygen species are known to contribute to progressive disease such as in ophthalmology, diabetes, kidney disease, cancer and neurodegenerative diseases.

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Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress

Cited by 13 publications

References 30 publications

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Mitochondrial Subtype Identification and Characterization

Non-invasive real-time imaging of reactive oxygen species (ROS) using multispectral auto-fluorescence imaging technique: a novel tool for redox biology

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