Purification and Culture of Encephalitozoon Spores

E. hellem and E. cuniculi spores were gifted by Dr. Han Bing from Shandong University, China, and cultured in RK13 cells, respectively. One week after infection, spores were purified from culture media by centrifugation in 70% Percoll (17089102, Cytiva, Marlborough, MA, USA) at 10,000× g for 15 min at room temperature (RT), then washed three times with sterile water, suspended in 0.5 mL of sterile distilled water, and stored at 4 °C until use [14 (link),40 (link),42 (link)]. The HEK293 and HFF cells were inoculated with spores by a cell-to-parasite ratio of 1:30, and all samplings of the infected cells were prepared 48 h post-infection (hpi).
An inhibitor of DRP1, Mdivi1 (ab144589, Abcam), was added to HFF cells at a 50 μM concentration for 4 h prior to infection, and then replaced the fresh complete medium, following a previous study [27 (link)], and it was added again after 24 hpi and treated for 24 h. Additionally, the same concentration of dimethyl sulfoxide (DMSO) (D8418, Sigma Aldrich) was added as a control.