Syto 61 red fluorescent nucleic acid stain

Uninfected and ring-infected RBCs were labelled with SYTO® 61 red fluorescent nucleic acid stain (Life Technologies) to detect parasite DNA. Briefly, 100 µL of RBCs were incubated in 1 mL of a 1/1000 dilution of SYTO61 in PBS/2 % AlbuMAX solution for 15 min. The RBCs were then washed three times with PBS and PKH26 (red) or PKH67 (green) lipophilic fluorescent cell linker probes were used to label HbAS and HbAA RBC membranes, respectively, according to the manufacturer’s instructions (Sigma-Aldrich, Saint-Quentin, France). Cell counting was performed using an Accuri C6 flow cytometer (Becton–Dickinson), and 20,000 events were recorded.

Heat-inactivated bacteria were labeled with SYTO61 Red Fluorescent Nucleic Acid Stain (2.5 µM in PBS; Life Technologies) at RT for 30 min and were afterwards washed three times in PBS. To analyze the Ca²⁺ dependency of the interaction, 50 µL labeled bacteria (3×10⁸ cells/mL) were incubated with 20 µg/mL Mincle-Fc diluted in lectin binding buffer or EDTA buffer at 4°C for 1 h. After three washing steps with PBS, Mincle-Fc binding to S. pneumoniae was detected with a PE-conjugated goat anti-hFc antibody (Dianova, Hamburg, Germany). Flow cytometric analysis was performed with a FACSCanto II flow cytometer (BD Pharmingen, Heidelberg, Germany). Data were analyzed using the FlowJo analysis software (Tree Star Inc., Ashland, OR, USA).