Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.
A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.
Thalassemia Center, Faculty of Medicine at Siriraj Hospital, Bangkok, Thailand.
K
K
Pattanapanyasat
K
K
Yongvanitchit
D G
DG
Heppner
P
P
Tongtawe
D E
DE
Kyle
H K
HK
Webster
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001710:Biotin / Q000737:chemistry* / Q000494:pharmacology; D002478:Cells, Cultured; D004912:Erythrocytes / Q000166:cytology / Q000469:parasitology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development*; D019809:Streptavidin
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate