Hepes

The 3D7 strain of P. falciparum was preserved at the Laboratory of Pathogen Infection and Immunity (Jiangnan University, Wuxi, China). The parasites were grown at 37 °C in a mixed environment of 90% N₂, 5% O₂, and 5% CO₂, and were maintained in RPMI Medium 1640 (Gibco, New York, USA) containing O⁺ human erythrocytes (4% hematocrit), HEPES (Meilunbio, Dalian, China), NaHCO₃ (Meilunbio, Dalian, China), AlbuMax Ⅱ (Sigma, San Francisco, CA, USA), hypoxanthine (Sigma, San Francisco, CA, USA), and gentamicin (Solarbio, Beijing, China).
The schizonts were purified by 60% percoll (Solarbio, Beijing, China) gradient centrifugation, and lysed in 0.1% saponin (diluted in PBS) for 5 min on ice with intermittent mixing. The lysed material was centrifuged at 15,000× g for 5 min and washed thrice with PBS. Then, the parasite lysate was collected and boiled in a SDS-PAGE sample loading buffer (Meilunbio, Dalian, China) for 7 min [13 (link)]. The total protein was separated on SDS-PAGE gel, and native PfSRA in the P. falciparum crude protein was captured by the anti-rPfSRA mice sera at 1:1000 dilution overnight and detected by HRP-conjugated goat anti-mouse IgG (CWBio, Beijing, China).

The cells were harvested and resuspended at 1 × 10⁶/ml in prewarmed DMEM (Life technologies, USA) containing 2% fetal calf serum (Kang Yuan Biology, China) and 10 mM HEPES (Dalian Meilun Biology Technology co., LTD, China). The cell suspension was gently mixed in 15 ml polypropylene centrifuge tube (Corning, USA). And then Hoechst 33342 (Invirtrogen, USA) was added to a final concentration of 5 ug/ml and verapamil (Sigma-Aldrich, USA) of 80 ug/ml to control group. The cells were incubated for 90 minutes at 37°C and mixed every 30 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, and resuspended in cold HBSS. Propidium iodide (Sigma-Aldrich,USA) was added into each tube at concentration of 1 ug/ml to discriminate dead cells. In order to avoid the effluxion of Hoechst 33342, further tests were performed at 4°C. SP cells were tested using flow cytometry and analyzed using Summit 5.2 software (Beckman Coulter, Inc., USA). The assays were conducted in triplicate and repeated at least 3 times.