Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes.
Enviado por Adelfa Serrano-Brizuela el
Título | Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes. |
Publication Type | Journal Article |
Year of Publication | 2011 |
Autores | Gerena, Y, Gonzalez-Pons, M, Serrano, AE |
Journal | Cytometry A |
Volume | 79 |
Issue | 11 |
Pagination | 965-72 |
Date Published | 2011 Nov |
ISSN | 1552-4930 |
Palabras clave | Animals, Bisbenzimidazole, Erythrocytes, Female, Flow Cytometry, Fluorescence, Fluorescent Dyes, Green Fluorescent Proteins, Limit of Detection, Malaria, Mice, Mice, Transgenic, Parasitemia, Plasmodium berghei, Propidium, Rhodamines, Ribonucleases, Staining and Labeling |
Abstract | Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. © 2011 International Society for Advancement of Cytometry. |
DOI | 10.1002/cyto.a.21119 |
Alternate Journal | Cytometry A |
PubMed ID | 22015734 |
PubMed Central ID | PMC3210507 |
Grant List | G12 RR003051 / RR / NCRR NIH HHS / United States G12 RR003051-27 / RR / NCRR NIH HHS / United States G12RR03051 / RR / NCRR NIH HHS / United States GM08224 / GM / NIGMS NIH HHS / United States P20 RR016470-06 / RR / NCRR NIH HHS / United States P20-RR016470 / RR / NCRR NIH HHS / United States R25 GM061838 / GM / NIGMS NIH HHS / United States R25 GM061838-10 / GM / NIGMS NIH HHS / United States R25-GM061838-10 / GM / NIGMS NIH HHS / United States S06 GM008224 / GM / NIGMS NIH HHS / United States S06 GM008224-23 / GM / NIGMS NIH HHS / United States |