Na 1.45 oil objective

CD8⁺ RTEs and MN T cells were stimulated as above and mitochondria and nuclear DNA were visualized using Mitotracker Red CMXRos (Thermo Fisher) and NucBlue Live ReadyProbes Reagent (Molecular Probes), respectively. Cells were allowed to settle onto poly-D-lysine (Sigma) coated glass-bottom petri dishes (MatTek) for 30 minutes before imaging. A Z-series with a 0.3 μm step size was collected at 37°C and 7% CO₂ using a Ti-E widefield microscope with a 100X NA 1.45 oil objective (Nikon), a solid-state light source (Spectra X, Lumencor), and a sCMOS camera (Zyla 5.5 Megapixel). Mitochondrial morphology was scored in a blinded fashion as “fragmented”: spherical mitochondria < 1.5 μm in diameter; “intermediate”: oblong and < 2.5 μm in length; and “reticular”: elongated and highly connected.

Spinogenesis experiments were performed as previously described^{5 (link)} with the exception that cells were treated on DIV20 and fixed 24 h after treatment on DIV21. The images were taken on a Nikon HCA Confocal microscope a with a 100x/NA 1.45 oil objective. DMSO and ketamine (10 μM) were used as vehicle and positive controls, respectively.

Spinogenesis experiments were performed as previously described^{14 (link)} with the exception that cells were treated on DIV19 and fixed 24 h after treatment on DIV20. The images were taken on a Nikon HCA Confocal microscope a with a 100x/NA 1.45 oil objective. DMSO and ketamine (10 μM) were used as vehicle and positive controls, respectively.

HEK293 (ThermoFisher) cells transfected with hACF7 GFP-EF1-EF2-GAR (residues 7018–7206, wild-type/mutant), GFP-EF1-EF2 (residues 7018–7116), or GFP-GST-EF1-EF2 (residues 7018–7116) constructs were fixed or imaged live 14–16 hrs post transfection. Cells were fixed with 4% paraformaldehyde in PHEM (5 mM HEPES, 60 mM PIPES pH 7.0, 10 mM EGTA, and 2 mM MgCl2) for 20 min. PBS-Triton (PBST)(0.5%) and PHEM-Triton (0.2%) supplemented with 1% normal bovine serum (Fisher Scientific) were used to permeabilize and block the cells, respectively. Antibodies were diluted in PHEM supplemented with 1% normal bovine serum. Antibodies used for immunofluorescence included a mouse monoclonal anti-α-tubulin antibody (clone DM1a, T6199; 1:500) (Sigma-Aldrich) and a Cy3-α-mouse conjugated secondary antibody (1:500) (Jackson ImmunoResearch Laboratories). Nuclei were stained with 4’, 6-diamidino-2-phenylindole (1:2000) (Molecular Probes, Invitrogen). Cells were mounted in fluorescence mounting medium (Dako). Images were acquired using an Eclipse Ti microscope with a 100x oil NA-1.45 objective (Nikon) by a Coolsnap HQ camera (Photometrics), driven by NIS Elements software (Nikon). Representative images were processed using Photoshop CS5 (Adobe Systems) and ImageJ (National Institutes of Health).