Dihydroethidium

Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously (Madeo et al., 1997 (link)). For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described (Madeo et al., 1997 (link)), and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD (Boehringer Mannheim). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (link)). The slides were rinsed with PBS and incubated with 0.3% H₂O₂ in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution (Boehringer Mannheim) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared.
Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine; Sigma Chemical Co.). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS^® Calibur (Becton Dickinson) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Free spin trap reagents N-tert-butyl-α−phenylnitrone (PBN; Sigma-Aldrich) and 3,3,5,5,-tetramethyl-pyrroline N-oxide (TMPO; Sigma-Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d.
To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated.

One microliter of whole blood was added to a tube containing 100 μl of PBS. Dihydroethidium (Sigma, Singapore), Hoechst 33342 (Sigma) and anti-CD45 coupled to allophycocyanine (APC) were added together to the blood sample. In preliminary experiments, we determined that the optimal doses for dihydroethidium and Hoechst 33342 were 5 μg/ml and 8 µM respectively using P. berghei-infected red blood cells (Figure S1). We also determined that the staining was stable over a 24 hours period (Figure S2). Rat IgG2a anti-mouse CD45 (clone 30F11.1, Miltenyi) or mouse IgG2a anti-human CD45 (clone 5B1, Miltenyi) monoclonal antibodies were used at a 1:50 dilution. In one set of experiments, Hoechst was substituted by SYBR Green I (Sigma, Singapore) at 0.25x dilution.
The diluted whole blood samples were incubated for 20 minutes at room temperature in the dark. After the incubation, 400 μl of cold PBS was added. The samples were acquired on an LSR II flow cytometer (Becton Dickinson, Singapore) using the UV laser (305 nm) to detect Hoechst 33342, the blue laser (488 nm) for GFP and Ethidium, and the red laser (633nm) for APC. In experiments using SYBR Green, samples were acquired with the Accuri C6 flow cytometer (Accuri cytometers Inc., Ann Arbor, MI) or LSR II flow cytometer (Becton Dickinson, Singapore). For samples with parasitemia less than 1%, 500,000 events were recorded, otherwise 100,000 events were recorded. FlowJo (Tree Star) was used for all flow cytometry analyses. In experiments using blood from infected mice, a negative control sample from a non-infected mouse was tested each day in parallel to define the threshold of positivity for the parasitemia.

HTM cells were processed as described above and then probed with dihydroethidium (DHE, S0063; Beyotime) at 37°C for 30 minutes, and the nuclei were stained with Hoechst 33342. After washing with PBS for three times, they were examined under the Leica fluorescence microscope.