Deltavision elite high resolution microscope system

TaC12 cells, T. gondii-infected HFF cells, and P. berghei-infected HeLa cells were grown on coverslips prior to fixation with 4% paraformaldehyde for 10 min at room temperature and were permeabilized by incubation in 0.2% Triton X-100 and blocking in 10% heat-inactivated FCS–phosphate-buffered saline (PBS). For analysis of TBL20, BL20, and Tpm803 cells, cytospins were prepared and fixed as described above. Antibodies were diluted in blocking solution, and DNA was stained with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). The coverslips were mounted using mounting medium (Dako). Samples were analyzed on a DeltaVision Elite high-resolution microscope system (GE Healthcare) with an Olympus IX-70 inverted microscope with a complementary metal oxide semiconductor (CMOS) camera, a 60× Olympus objective, and SoftWorx (Applied Precision) software. The samples shown in Fig. 2B (see also Movie S1 and S2 in the supplemental material) were analyzed on a DeltaVision OMX Blaze system (GE Healthcare) with scientific complementary metal oxide semiconductor (sCMOS) cameras at the Biozentrum in Basel, Switzerland. Images were processed by using Fiji (ImageJ) software and Photoshop (Adobe). Time-lapse imaging was performed using a DeltaVision Elite high-resolution microscope system (GE Healthcare) (movie S3) or an LSM 5 Duo microscope system (Zeiss) (Movie S4).

Cells were seeded and grown on coverslips and fixed with 4% PFA for 10 min at room temperature. Cells were permeabilised in 0.2% Triton X‐100 and antibodies were diluted in 10% heat‐inactivated FCS in PBS. DNA was stained with DAPI (Invitrogen), and samples were mounted by using mounting media (DAKO). Fixed samples and time‐lapse experiments were analysed on a DeltaVision Elite High Resolution Microscope system (GE Healthcare) with Olympus IX‐70 inverted microscope with a CMOS camera, 60× Olympus Objective, and SoftWorx (Applied Precision) software. Samples in Figures 1, 2 (top and bottom panels), S1B,C, and S2A,D were analysed on a Nikon Eclipse 80i wide‐field microscope with a Hamamatsu Orca R2 camera using PlanApo objectives (Nikon) and the OpenLab 5 software (Improvision). Samples in Figure S9 were analysed on the DeltaVision OMX Blaze system (GE Healthcare) with sCMOS cameras at the Biozentrum in Basel, Switzerland. Fiji (ImageJ) software (Schindelin et al., 2012) and Photoshop (Adobe) were used to process the images.

Cells were grown on coverslips, treated for 18 h with the indicated drugs, and fixed with 4% formalin for 10 min at room temperature. Cells were permeabilized in 0.2% Triton X‐100, and antibodies, diluted in 10% heat‐inactivated FCS in PBS, were applied for 1 h at room temperature. DNA was stained with DAPI (Invitrogen) before mounting using DAKO mounting media. Analysis was performed on a DeltaVision Elite High Resolution Microscope system (GE Healthcare) with Olympus IX‐70 inverted microscope with a CMOS camera, 100× Olympus Objective, and softworx (Applied Precision, Issaquah, WA, USA) software. Per condition, four times 16 adjacent image fields were randomly taken for quantification. fiji software was used to process the images (Schindelin et al., 2012).