Establishment of optimal SYBR Green I staining for fluorescence assay

To determine the optimal concentration of SYBR Green I and the optimal incubation time for discriminating between infected and noninfected RBCs with minimal background noise from WBCs, ten female BALB/c mice were used in three separate experiments. The mice were divided equally into two groups. The first group was inoculated intraperitoneally with 1 × 10⁷ B. microti-infected RBCs, while the second group remained uninfected and was used as a blank control. SYBR Green I concentrations ranging from 0.25 × to 8 × were used to stain RBCs from B. microti-infected mice at 2.5% HCT. The levels of parasitemia in the infected mice were monitored daily by microscopic examination of Giemsa-stained thin blood smears prepared from venous tail blood to determine the days with peak parasitemia. On days showing peak parasitemia, fluorescence analysis was performed after the samples were incubated in the dark at room temperature for 1, 2, 3, 4, 5, and 6 hours. Next, the experiment was repeated in another ten female BALB/c mice divided equally into 2 groups. The first group remained without infection and was used as a negative control, while the second group was intraperitoneally inoculated with 1 × 10⁷ B. microti-infected RBCs and used as a positive control. The emitted fluorescence signals were monitored every two days until the cessation of parasitemia by the Babesia fluorescence–based assay using 1x, 2x, 4x, and 8x SG I nucleic acid stain at 2.5 % HCT and compared with the levels of parasitemia in infected mice as detected by the microscopy method through the examination of 1000 erythrocytes in Giemsa-stained thin blood smears prepared from venous tail blood. The plates were incubated for 1 hour, and fluorescence values were determined as formerly described. The experiment was repeated two times.