35 mm live cell imaging dishes

CHO cells were transfected with mCherry-ER and YFP-AT or YFP-AAT constructs in 35 mm live-cell imaging dishes (MatTek) and, 24 hours later, imaged in tissue culture medium supplemented with 25 mM HEPES, pH 7.0 to 7.6. Live-cell imaging was performed as reported previously (33 (link), 62 (link)). Confocal micrographs were acquired using a Zeiss LSM780 laser scanning confocal microscope (Zeiss) using a ×63 1.4NA oil immersion objective. YFP- and mCherry-tagged proteins were excited at 514 nm and 561 nm, respectively, and images were captured with 1,028 × 1,028 pixel frame size.

CHO cells were grown in 35 mm live cell imaging dishes (MatTek, Ashland, MA, USA), transfected as required, and left in culture for 24 h after transfection before imaging. Cells were imaged in tissue culture medium supplemented with 25 mM HEPES, pH 7.0 to 7.6. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) imaging was undertaken using a confocal laser scanning microscope (LSM710; Carl Zeiss GmbH, Jena, Germany) in a 37°C/5% v/v CO₂ chamber with a ×40/1.3 NA oil objective. Zen 2010 software was used for all imaging. A small region of interest was photobleached using laser powers to achieve an approximately 50% bleach (variable by protein) using the 488 nm (GFP), 514 nm (YFP), and/or 594 nm (mCherry) lasers as necessary. Fluorescence loss or recovery was monitored with a 0.1 ms delay between images. Data from a minimum of 12 cells per condition were normalized to reference regions of interest. Confirmation of bleaching in the z plane was confirmed using a postbleach z stack. For 3-dimensional imaging, z stacks were taken using overlapping confocal slices, and images were reconstructed into 3-dimensional movies using Imaris software (Bitplane, Zurich, Switzerland).