Abstract
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.
Highlights
Malaria remains a major cause of morbidity and mortality worldwide
Optimization of optical filters allowed for the detection of 50-150% more green fluorescent protein (GFP)-expressing parasites as compared to use of filters supplied with the flow cytometer, and sorted populations were >90% pure
In vitro culture of Plasmodium falciparum parasites and preparation of cells prior to flow cytometry analysis Three P. falciparum lines were mainly used in this study: a non-fluorescent gametocyte-producing clone termed P2G12 derived from the reference strain 3D7 (P2G12 Wild type (WT), [9]); and two transgenic lines derived from this clone which express GFP (164/GFP, [9]), and tandem dimer tomato fluorescent protein (164/Tandem dimer Tomato fluorescent protein (tdT), [11]), respectively, from the gametocyte-specific promoter of the gene PF10_0164
Summary
Malaria 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. The major aim of this study was to optimize filter sets in order to improve the detection of GFP-expressing transgenic parasites Another hurdle must be overcome when sorting rare populations, which is essential for examination of the malaria life cycle in vivo: how to obtain sufficient cell numbers while ensuring high purity of the sorted cell population. Optimization of optical filters allowed for the detection of 50-150% more GFP-expressing parasites as compared to use of filters supplied with the flow cytometer, and sorted populations were >90% pure This can advance Plasmodium gametocyte research, since the challenge in isolating these parasite stages often lies in either low overall number of gametocytes in certain parasite lines or in contamination of rare desired events (e.g. very young gametocytes or mature gametocytes out of a mixed population) with unwanted parasite stages (e.g. asexual stages). Based on the approaches described here, investigators will be able to rapidly evaluate optical filter efficiency using cell number and desired purity for a given level of statistical significance
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