Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

Abstract

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono-and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. ' 2011 International Society for Advancement of Cytometry Key termsPlasmodium falciparum; melatonin; cell cycle; flow cytometry MALARIA is caused by the Apicomplexan parasite Plasmodium falciparum. Its asexual replicative cycle inside red blood cell (RBC) is responsible for pathogenesis and induces several structural and biochemical changes within the host cell (1,2). One striking feature regarding P. falciparum infection is associated with 48-h fever peaks intervals, as a result of from synchronous release of merozoites into the blood stream (3).Melatonin, a hormone produced by the pineal gland of vertebrates, transmit the darkness signal based on circadian and seasonal time measurements (4). The presence of melatonin, however, is not restricted to vertebrates but is also found in phylogenetically divergent species including bacteria, plants and protozoa (4). We have previously shown that this hormone is able to synchronize Plasmodium falciparum and P. chabaudi cell infections (5-7). The synchronicity was lost in vitro when parasites had been incubated with the melatonin antagonist luzindole; the same effect was obtained in vivo in pinealectomized mice and after the injection of luzindole (5). In P. falciparum, melatonin induces a complex signaling pathway with the participation of IP3 generation (8) and subsequent transients in cytosolic calcium concentration, cAMP production and protein kinase A (PKA) (9,10) and protease activation (11).Flow cytometry (FCM) is a powerful method for evaluation of human erythrocyte infection rates as well as for discrimination of Plasmodium falciparum developmental stages (12-16). Several methods of detection of malaria parasite by flow cytometry have been developed taking advantage of the absence of DNA in erythrocytes. Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia

The Structurally Related Auxin and Melatonin Tryptophan‐Derivatives and their Roles in Arabidopsis thaliana and in the Human Malaria Parasite Plasmodium falciparum

“…The stage distribution and parasitemia were assessed by flow cytometry according to the protocol established by Schuck et al. ().…”

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid

“…Parasite stages were standardised with synchronised cultures according to previously published methods (Schuck et al . ). Concentration of 4a and 4b to inhibit parasite growth was determined comparing the fluorescence incorporated into the treated culture and untreated culture (control).…”

“…Parasitemia and proportions of parasites at each stage were determined from dot plots [side scatter (SSC) vs. fluorescence] of 10 5 cells acquired on an FC using CELLQUEST (Becton & Dickinson) and FlowJo software (Tree Star Inc., version 8). Parasite stages were standardised with synchronised cultures according to previously published methods (Schuck et al 2011). Concentration of 4a and 4b to inhibit parasite growth was determined comparing the fluorescence incorporated into the treated culture and untreated culture (control).…”

The GCaMP3 – A GFP-based calcium sensor for imaging calcium dynamics in the human malaria parasite Plasmodium falciparum

“…The calcium response was determined from dot plots [side scatter (SSC) versus fluorescence] of 10 5 cells acquired on a FACS Calibur flow cytometer using CELLQUEST software (Becton & Dickinson). GCaMP3 was excited with a 488 nm argon laser and the fluorescence emission was collected at 520–530 nm [8].…”