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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Ivan A Vorobjev, Kathrin Buchholz, Prashant Prabhat, Kenneth Ketman, Elizabeth S Egan, Matthias Marti, Manoj T Duraisingh, Natasha S Barteneva
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-312) contains supplementary material, which is available to authorized users.
Ivan A Vorobjev, Kathrin Buchholz contributed equally to this work.

Competing interests

PP is employed by Semrock Inc. Other authors do not have any competing interests.

Authors’ contributions

The work was carried out in collaboration between all authors. IAV and KB designed experiments, carried out the laboratory experiments, analysed the data, interpreted the results and wrote the draft. KK, PP and EE co-designed and performed experiments and contributed in data interpretation and report writing. MM and MD supervised KB and EE, coordinated the research, and commented on the manuscript. NSB designed and coordinated research, analysed the data, wrote the manuscript, and provided funding. All authors have contributed to, seen, and approved the final manuscript.

Abstract

Background

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. 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.

Methods

Detection 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.

Results

A 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.

Discussion

Filter 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.
Zusatzmaterial
Additional file 1: Spectral characteristics of the optical filters used in the study (A) and the transmission spectra of three different optical filters used for the detection of GFP-positive events superimposing the GFP emission spectrum (B). (TIFF 594 KB)
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Additional file 2: Relative yield (%) of GFP + gametocytes (line 164/GFP) collected with a FACSAria cytometer, equipped with dichroic filter 466LP vs a FACSAria cytometer equipped with 502LP dichroic and different bandpass filters. (TIFF 5 MB)
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Additional file 3: Representative image gallery of sorted red blood cells infected with GFP-expressing parasites (line 164/GFP). The parasites were sorted using a 517/20 filter (dichroic 502LP) and images were acquired with Imagestream 100 imaging cytometer. Left channel: bright field, middle channel: fluorescent channel 3 (GFP-channel), right channel: merged bright field and green channel. (TIFF 11 MB)
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Additional file 4: Cytometer and optical filter information available from publications on malaria research. (DOC 53 KB)
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Additional file 5: List of optical filters interchangeability in some cytometers. (DOC 48 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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