Brdu red

Tightly synchronized ring-stage parasites (NF54) were fixed for 30 min in freshly prepared fixative (4% paraformaldehyde and 0.005% glutaraldehyde). After fixation, cells were rinsed three times with phosphate-buffered saline (PBS) and incubated with permeabilization solution (0.1% Triton X-100 in PBS) for 10 min on ice. The cells were washed twice with PBS and one time with wash buffer supplied with TUNEL assay kit bromodeoxyuridine (BrdU) red (Abcam; catalogue number ab6610). TUNEL assay was performed as per manufacturer guidelines. Briefly, following a washing, 50 μl of TUNEL reaction mixture (DNA labeling solution) was added to each sample. The cells were incubated for 60 min at 37°C with intermittent shaking. The cells were then washed three times with rinse buffer (5 min each time) and resuspended in 100 μl of antibody solution for 30 min at room temperature. The cells were then washed three times with PBS, mounted using Invitrogen Molecular Probes ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI), and imaged using a Nikon Eclipse Ti-E microscope equipped with a CoolSNAPMyo charge-coupled-device (CCD) camera.

To determine cell death several methods were used. LDH cytotoxicity assay (ThermoFisher Scientific) and TUNEL assay (BrDU-Red; ab66110; Abcam) were carried out according to the manufacturer’s protocol.
Determination of bioluminescence (BLI) loss was used as a marker for cell death in K562-fLUC cells. After appropriate treatment, 500 μM D-luciferin (Xenogen) was added to cells. BLI was measured using IVIS® Lumina Series III (Perkin Elmer). Data was analyzed with Living Image® 4.5.2 (Perkin Elmer). From Average Radiance values (ARV) the percentage of specific lysis was calculated using formula: % specific lysis = 100*(spontaneous death ARV-test ARV)/(spontaneous death ARV-maximal killing ARV).

TUNEL staining was applied to apoptotic rate detection in myocardial tissues. For TUNEL staining, we stained myocardial tissue slides with TUNEL Assay kit (BrdU-Red, ab66110; Abcam, Cambridge, UK) following the procedure instructions and the fluorescence images were captured by using Fluorescence microscope. At the same time, we stained cardiomyocyte in tissue slides with Desmin antibody (Alexa Fluor 555 Anti-Desmin antibody, ab203422; Abcam, Cambridge, UK shown in green) at 1/100 dilution by immunofluorescence staining assay. Moreover, the nuclei in tissue slides were stained by using DAPI (ab285390; Abcam, Cambridge, UK). DAPI was used for nuclei stain at concentration of 10 μg/ml under room temperature for 15 min avoid of light.