FCS experiments were carried out on an LSM710 microscope equipped with a ConfoCor3 extension and a 40× water objective (NA = 1.2). A 633 nm laser was used for excitation together with a BP655-710nm emission filter. Measurements were conducted in 8-well chambered cover glass slides (Nunc LabTek). Alexa 633 dye (A33084, Molecular Probes) and Atto 633 labeled TelC ALT-FISH probe were diluted to a final concentration of 20 nM in 10 mM Tris–HCl pH 8, 10 mM NaCl, 0.002% Tween-20. Tetraspeck fluorescent bead 0.1 μm standards (Thermo Fisher Scientific, T7279) were used undiluted as provided by the manufacturer at ∼300 pM concentration. FCS data were fitted with the ZEN 2008 software using a single component fit.
8 well chambered cover glass slides
Manufactured by Thermo Fisher Scientific
The 8-well chambered cover glass slides are a laboratory equipment designed for cell culture applications. They provide a transparent, multi-compartment platform for microscopic observation and analysis of cells in a controlled environment.
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Lab products found in correlation
2 protocols using 8 well chambered cover glass slides
1
FCS Microscopy of Alexa 633 and Atto 633 Probes
2
Live-cell FRET and cell death analysis
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Time-lapse microscopy movies were recorded with a Deltavision fluorescence microscope equipped with an environmental chamber (Olympus, Applied Precision) at 10x magnification with frames every 3 minutes for 24 hours. Cells grown in 8-well chambered cover glass slides (Nunc) were shifted into phenol red-free medium (Invitrogen) supplemented with 10% fetal bovine serum and L-glutamine for imaging. For FRET signal analysis, ratio of background-subtracted CFP and YFP images were created by using ImageJ and custom plug-ins23 (link). Signals were normalized by subtracting the minimum value across all time points from each single-cell time course. IMS-RP release in cell cytoplasm was analyzed in ImageJ by visual inspection, which enabled an identification of the first frame of IMS-RFP release and subsequent time of cell death of each single-cell of the cell population analyzed.
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