The principle of pLDH assay is shown in Figure 12a. Red blood cell (RBC) type O+ was obtained from the local Red Cross. P. falciparum 3D7 was maintained in a RPMI-1640 medium (supplemented with Albumax II (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and contained RBC (3% hematocrit). P. falciparum cell was synchronized into the ring form stage using 5% sorbitol, and then adjusted so the parasitemia was 0.3%. The sample was added to 96-well microplate, followed by the addition of 100 μL of the P. falciparum cell culture. The cell was incubated for 3 days at 37 °C, 5% CO2, 5% O2. Cold PBS was added to the cell culture, as much as 200 μL, then centrifuged at 1700× g for 10 min. After discarding the supernatant, 100 µL of LDH reaction mixture (100 mM Tris-HCl pH 8.0, 50 mM sodium-L-lactate, 0.25% (v/v) Triton X-100), 0.2% nitro blue tetrazolium (NBT), 50 µg/mL 3-acetyl pyridine NAD (APAD), and 0.05 U diaphorase) was added into each well and incubated at 37 °C for 30 min before the absorbance was measured at 650 nm using a microplate reader. Inhibition activity was calculated using formula in Figure 12b. Cell cultures with the addition of DMSO (final concentration 0.4%) and atovaquone (final concentration 1 µM) were regarded as negative and positive controls, respectively.
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