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.
PP is employed by Semrock Inc. Other authors do not have any competing interests.
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.
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.
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.
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.
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.
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)12936_2012_2571_MOESM1_ESM.tiff
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)12936_2012_2571_MOESM2_ESM.tiff
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)12936_2012_2571_MOESM3_ESM.tiff
Additional file 4: Cytometer and optical filter information available from publications on malaria research. (DOC 53 KB)12936_2012_2571_MOESM4_ESM.doc
Additional file 5: List of optical filters interchangeability in some cytometers. (DOC 48 KB)12936_2012_2571_MOESM5_ESM.doc
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Mota MM, Thathy V, Nussenzweig RS, Nussenzweig V: Gene targeting in the rodent malaria parasite Plasmodium yoelii. Mol Biochem Parasitol. 2011, 113: 271-278. CrossRef
Miambo G, Kumar N: Transgenic rodent Plasmodium berghei parasites as tools for assessment of functional immunogenicity and optimization of human malaria vaccines. Eukaryot Cell. 2008, 7: 1875-1879. 10.1128/EC.00242-08. CrossRef
Janse CJ, Franke-Fayard B, Waters AP: Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc. 2006, 1: 620-623.
Aingaran M, Zhang R, Law SK, Peng Z, Undisz A, Meyer E, Diez-Silva M, Burke TA, Spielmann T, Lim CT, Suresh S, Dao M, Marti M: Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum. Cell Microbiol. 2012, 14: 983-993. 10.1111/j.1462-5822.2012.01786.x. PubMedCentralCrossRefPubMed
Prabhat P, Erdogan T: Measurement of optical filter spectra. 2010, [ http://www.semrock.com]
Spectra of various fluorescent proteins. [ http://www.tsienlab.ucsd.edu]
Miao J, Li X, Cui L: Cloning of Plasmodium falciparum by cell sorting. Exp Parasitology. 2010, 126: 198-202. 10.1016/j.exppara.2010.04.022. CrossRef
Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 2004, 22: 1567-1572. 10.1038/nbt1037. CrossRef
Ramanujan N: Fluorescence spectroscopy of neoplastic and non-neoplastic tissues. Neoplasia. 2000, 2: 89-117. 10.1038/sj.neo.7900077. CrossRef
Pearse AG: Histochemistry: theoretical and applied. 1972, NY: Williams & Wilkins Publishers, 3
Golan A, Kerem Z, Tun OM, Luzzatto T, Lipsky A, Yedidia I: Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectbacterium cartovorum ssp. cartovorum in Ornithogalum dubium plantlets. J Appl Microbiol. 2007, 108: 1136-1144. CrossRef
Zimmerlin L, Donnenberg VS, Donnenberg AD: Rare event detection and analysis in flow cytometry: bone marrow mesenchymal stem cells, breast cancer stem/progenitor cells in malignant effusions, and pericytes in disaggregated adipose tissue. Methods Mol Biol. 2011, 699: 251-273. 10.1007/978-1-61737-950-5_12. CrossRefPubMed
- Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters
Ivan A Vorobjev
Elizabeth S Egan
Manoj T Duraisingh
Natasha S Barteneva
- BioMed Central
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