Biotek lionheart fx

T cells were labeled with the calcium indicator Fluo-4 AM (Thermo Fisher). A total of 10⁶ cells in 1 mL of RPMI 1640 medium containing 10% FBS were loaded with 5 mM Fluo-4 AM for 20 min at 37°C in the presence of 0.1% Pluronic F-127 and 0.25 mM Probenecid (Thermo Fisher). The cells were washed three times with RPMI 1640 and incubated at 37°C for an additional 5 min. The cells were then resuspended in RPMI 1640 medium containing 10% FBS, and their Ca²⁺ levels were assessed using a CytoFLEX Flow Cytometer (Beckman Coulter). For live-cell microscopy, loaded T cells were plated on poly-L-lysine-coated culture plates, centrifuged at 100 g for 2 min, and placed in an incubator at 37°C for 30 min. Images were acquired using a BioTek-lionheart FX (BioTek).

Cells at passage 5 were digested and resuspended in the medium described above and seeded in a 96-well plate at a density of 1 × 10⁴ cells per well (passage 6). The plates were incubated in BioTek-lionheart FX (BioTek, USA), and the cultured live cells were continuously monitored for 72 h at 37 °C in a 5% CO₂ atmosphere. Images were captured every 60 min using a × 4 objective, and the cellular numbers were directly counted by Gen5 software (four replicates per sample). In addition, 5-ethynyl-2′-deoxyuridine (EdU, Thermo Fisher, USA) staining was used to confirm cellular proliferation. Briefly, cells were seeded in 48-well plates at a density of 2 × 10⁴ cells per well (passage 6) in a medium containing 10 μM EdU and incubated for 24 h. Thereafter, the cells were fixed, permeabilized and stained with reaction cocktail for 1 h at 37 °C according to the manufacturer’s protocol. Proliferating cells were identified by red staining in the nucleus, and total cells were stained with DAPI (4′, 6-diamidino-2-phenylindole, blue, Thermo Fisher).

Initially, the medium in which the cerebral organoids was maintained was removed, and pre-chilled Cell Recovery solution (Corning) was added. Using a wide-bore tip, the solution was pipetted up and down, and the Matrigel was carefully removed to avoid damaging the organoids. Then, the cell culture plates were incubated at 4°C for 20 min. When the organoids were free from the Matrigel, they were washed three times with PBS and then fresh media were added. Next, 1 × 10³ ME49 tachyzoites and RH tachyzoites were incubated with separate cerebral organoids, each in a well with 200 μl of culture medium, in a 96-well plate for 4 h at 37°C in a humidified incubator with an atmosphere of 5% CO₂. After 4 h, the cerebral organoids were washed three times with PBS and fresh medium was added.
For live-cell imaging, GFP-tagged T. gondii-infected organoids were transferred to a 35-mm dish, and fresh medium was added. The organoids were maintained at 37°C in an automated microscope chamber with a humidified atmosphere of 5% CO₂ (BioTek Lionheart FX, BioTek Instruments, Winooski, VT, USA). Time-lapse images were acquired every 30 min for a total of 50 h. In total, nine positions were imaged, and a z-stacking process was performed.