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

Ivan A. Vorobjev, Kathrin Buchholz, Prashant Prabhat, Kenneth Ketman, Elizabeth S. Egan, Matthias Marti, Manoj T. Duraisingh, Natasha S. Barteneva

Research output: Contribution to journalArticle

10 Citations (Scopus)

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.

Original languageEnglish
Article number312
JournalMalaria Journal
Volume11
DOIs
Publication statusPublished - 2012
Externally publishedYes

Fingerprint

Plasmodium
Parasites
Green Fluorescent Proteins
Erythrocytes
Malaria
Microscopy
Flow Cytometry
Parasitemia
Gene Expression Profiling
Plasmodium falciparum
Population
Proteins
Morbidity

Keywords

  • Cell sorting
  • Fluorescent proteins
  • Malaria
  • Optical filter
  • Plasmodium
  • Rare cells

ASJC Scopus subject areas

  • Infectious Diseases
  • Parasitology

Cite this

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters. / Vorobjev, Ivan A.; Buchholz, Kathrin; Prabhat, Prashant; Ketman, Kenneth; Egan, Elizabeth S.; Marti, Matthias; Duraisingh, Manoj T.; Barteneva, Natasha S.

In: Malaria Journal, Vol. 11, 312, 2012.

Research output: Contribution to journalArticle

Vorobjev, Ivan A. ; Buchholz, Kathrin ; Prabhat, Prashant ; Ketman, Kenneth ; Egan, Elizabeth S. ; Marti, Matthias ; Duraisingh, Manoj T. ; Barteneva, Natasha S. / Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters. In: Malaria Journal. 2012 ; Vol. 11.
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AU - Egan, Elizabeth S.

AU - Marti, Matthias

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AB - 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.

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