Applied precision deltavision microscope system

1 × 10⁶ Hepa 1-6 hepatocyte cells resuspended in DMEM (Sigma, St. Louis, MO, USA) with 10% v/v FCS (Sigma) were plated in 40-mm glass-bottom culture dishes (Greiner bio-one, Monroe, NC, USA, at 5 × 10⁵ cells/well) for at least 4 h before inoculation with 1 × 10⁴ freshly isolated or cryopreserved P. berghei sporozoites. The infected cultures were washed twice with PBS and imaged at 36 h post-infection (p.i.) (Applied Precision DeltaVision Microscope System, GE Healthcare, Issaquah, WA, USA) [27 (link)]. Cryopreserved Pb-Luc-GFP parasites were visualized using the GFP channel to assess conservation of transgenic properties following cryopreservation.
For in vivo infections, 1 × 10³ freshly isolated or cryopreserved P. berghei or P. yoelli sporozoites were enumerated and resuspended in 200 µL complete normal saline before inoculating into B6 mice intravenously (i.v.). The parasite burdens or immune responses were assessed at 24 h, 44 h, or 7–10 days p.i.

HMI-9 medium (5–10 mL) containing 1 × 10⁵–2 × 10⁶cells/mL of T. brucei was centrifuged (5 min, 3000 × g at room temperature) to pellet the cells. After supernatant removal cells were resuspended in 1 mL of PBS containing 4% paraformaldehyde (Affymetrix; Santa Clara, CA) and incubated for either 1 min at room temperature or up to two weeks at 4 °C. Fixed cells were adhered to poly-L-lysine coated coverslips for 15 min at room temperature. Coverslips were washed with approximately 0.1–1 mL of PBS, air dried, then mounted on 2 μL of DAPI (1.5 μM) in Vectashield (Vector Labs; Burlingame, CA). For quantitation, 150 cells per sample were counted for each independent experiment using an EVOS^® FL inverted fluorescence microscope (Life Technologies; Grand Island, NY). Images for figures were captured on an Applied Precision DeltaVision microscope system (GE Healthcare; Issaquah, WA) using an Olympus IX-71 inverted microscope (Olympus; Center Valley, PA) at 60X, ex: 435 nm, em: 448 nm. To compare effects on organelle DNA duplication by drug treatment and vehicle control groups, Student’s t-test was performed in Excel to generate p-values.