Coolsnap hq monochrome ccd camera

B7-negative (ProAdICAM-1) and B7-positive (ProAdICAM-1/B7-1) transfectants [11] (link) were pre-incubated with or without 2.0 µg/ml of OVA peptide for 1 hour at 37°C, mixed with T cells at a 1∶1 ratio, and centrifuged at Rcf 2000 for 20 seconds at RT. The cell pellet was incubated for 5 minutes at 37°C. T cells were resuspended in 200 µl DMEM, plated on poly-L-lysine (Sigma, St. Louis, MO) coated coverslips for 3 minutes at 37°C, and fixed in 3% (w/v) paraformaldehyde. Samples were imaged at room temperature in Mowiol-DAPCO on a Zeiss Axiovert microscope with a 63×1.4 NA on a Plan-Apochromat oil immersion objective. Images were collected utilizing a CoolSNAP HQ monochrome CCD camera (Roper Scientific). Nearest-Neighbor deconvolution and digital analysis were performed using SlideBook software (Intelligent Imaging Innovations).

To visualize mitochondria and detect ΔΨ changes, myotubes were loaded with 30 nM tetramethylrhodamine methyl ester (TMRM, Invitrogen) in DMEM-HG differentiation medium for exactly 25 minutes. Under these conditions TMRM operated in non-quenching mode. Then cells were washed and transferred to a HEPES-Tris (HT) medium (132 mM NaCl, 4.2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5.5 mM D-glucose and 10 mM HEPES, pH 7.4). The coverslips were mounted in an incubation chamber and placed on the stage of an inverted microscope (Axiovert 200 M, Carl Zeiss, Germany). For noise analysis, empty coverslips were mounted on the microscope and HT-buffer containing different concentrations of TMRM (0–80 nM) was used. TMRM was excited at 540 nm using a monochromator (Polychrome IV, TILL Photonics, Gräfelfing, Germany) and a Zeiss 40×/1.3 NA Plan NeoFluar objective. Fluorescence light was directed using a 560DRLP dichroic mirror (Omega Optical Inc, Brattleboro, VT, USA) and a 565ALP emission filter (Omega Optical Inc.) onto a CoolSNAP HQ monochrome CCD-camera (Roper Scientific, Vianen, The Netherlands). The microscopy hardware was controlled using Metafluor 6.0 software (Universal Imaging Corporation, Downingtown, PA, USA) and images were recorded every 2 seconds for a period of 20 minutes using a 300 ms image acquisition time.