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.
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.
