High-throughput methods for evaluation of in vivo efficacy of candidate compounds against Plasmodium parasites are necessary during the
antimalarial drug development process. It is essential that enumeration of
parasitemia in the infected blood from experimental host animals is accurate and reliable. Flow cytometric enumeration of parasitized cells stained with
fluorescent dye is a rapid alternative method to conventional microscopic counting. In this study, a protocol for flow cytometric enumeration of rodent
malaria parasite Plasmodium berghei-infected red blood cells (RBC) stained with
SYBR Green I was developed. The optimal concentration of
SYBR Green I used to
stain infected RBC was 4× for 30 min. This
SYBR Green I staining protocol in combination with the bi-dimensional FL-1(530)/FL-3(620) detection method accurately detects
parasitemia above 0.02%. The
dye is stable during the prolonged incubation period necessary for accurate enumeration of
parasitemia, with no loss of fluorescent signal over a period of hours. This protocol was validated in an
antimalarial assay and the result was comparable to that obtained from conventional microscopic counting. The
SYBR Green I flow cytometric protocol is thus a rapid and precise tool for high-throughput in vivo
antimalarial drug screening.