Coolsnap hq ccd camera

Cell migration chambers (Millicell EZ slide eight-well glass, Millipore) were prepared by coating with Protein A (Invitrogen, 20 μg/mL), ICAM-1 (2.75 μg/mL), CXCL10 and CXCL12 (400 ng/mL) in PBS. 5x10⁴ PD1-sorted CD8⁺ T cells were plated in Leibovitz’s medium supplemented with 2 mg/mL D-glucose in a 37°C chamber. Video microscopy was conducted using a TE2000-U microscope (Nikon) coupled to a CoolSNAP HQ CCD camera with a 10x objective and 0.45 numerical aperture. T Cells and cancer cells were stained with CellTrace CFSE, CellTrace Violet, or CellTrace FarRed per manufacturers protocol (ThermoFisher). T cells were plated in L-15 medium at least 20 min at 37°C before imaging. For T cells alone, bright field or DIC images were acquired every 15 sec for 20 min. For T cell and cancer cell coculture movies, the stained cancer cells were plated 24 hours before the start of imaging. Bright field and fluorescent images were captured every 30 seconds for at least 2 hours.

Cell migration chambers (Millicell EZ slide eight-well glass, Millipore) were prepared by coating with Protein A (20 μg/mL), ICAM-1 (2.75 μg/mL), and CXCL12 (400 ng/mL) in PBS. Day 4 activated CD8⁺ T cells were plated in L-15 medium (Invitrogen, Leibovitz’s medium + 2 mg/mL D-glucose) in a 37°C chamber. Video microscopy was conducted using a TE2000-U microscope (Nikon) coupled to a CoolSNAP HQ CCD camera with a 10x objective and 0.45 numerical aperture. T cells were plated in L-15 medium at least 20 minutes at 37°C before imaging. Treatment of cells with inhibitors before imaging: CoCl₂ treatment was overnight (~16 hours) and maintained in L-15 media; FCCP and oligomycin were added to the cells 20 minutes before the movie started when cells are plated. OptoMito-On and Mock or GFP T cell migration movies all received 500 nm illumination with a Texas Red filter. Bright field or DIC images were acquired every 15 seconds for 20 minutes.

Peritoneal macrophages derived from Ip6k1^+/+ and Ip6k1^−/− mice grown on glass-bottom dishes were serum-starved for 1 h and incubated with 750 nm carboxylated latex beads (07759, Polysciences) for 1 h to allow phagocytosis. The unphagocytosed beads were washed with serum-containing media, and the cells were incubated for 1 h in the same medium at 37°C. Differential interference contrast (DIC) images were acquired using a live cell multipoint imaging system (Nikon Ti Eclipse) equipped with a ×100 1.4 N.A. objective, ×1.5 intermediate magnification, and a CoolSnap HQ CCD camera. Images were analyzed by ImageJ to identify the cell and nuclear boundaries and to examine the movement of phagocytosed beads along the long edge of the cell towards the nucleus. The fractional distance of each bead from the nuclear centroid was calculated as the ratio of the distance of the bead from the center of the nucleus to the distance of the cell membrane from the center of the nucleus along the line joining the centroid and bead.