For fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) experiments, 2 × 104cells were seeded on 8-well Lab-Tek chambered cover glass (Nunc). To visualize spots, cells were transfected 1 h after cell seeding with a plasmid encoding OR3-EGFP using FuGENE HD transfection reagent (Promega) according to the manufacturer’s instructions for a 3:1 transfection reagent:DNA ratio. To express free mEGFP as a control for FCS, cells were transfected 1 h after cell seeding with a plasmid encoding mEGFP (kindly provided by J. Lippincott-Schwartz) using FuGene6 transfection reagent (Promega) according to the manufacturer’s instructions for a 3:1 transfection reagent:DNA ratio. FCS and FRAP experiments were performed 24–48 h after cell seeding/transfection in CO2-independent imaging medium (Gibco, custom-made) supplemented with 20% (v/v) fetal bovine serum, 1% (v/v) sodium pyruvate, and 1% (v/v) L-glutamine.
Co2 independent imaging medium
Manufactured by Thermo Fisher Scientific
CO2-independent imaging medium is designed to facilitate cell imaging in the absence of a CO2-controlled environment. It maintains an optimal pH for cell growth and imaging without the need for a CO2 incubator.
Automatically generated - may contain errors
2 protocols using co2 independent imaging medium
1
Fluorescence Microscopy Imaging Protocols
2
Live-cell Imaging of Mitosis
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Culturing medium was removed from transfected cells plated on poly-d -lysine–coated filming dishes (MatTek) and replaced with CO2-independent imaging medium (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin. Using the 40× objective with differential interference contrast (DIC) and GFP fluorescent imaging, 10 randomly selected XY fields were programmed for imaging every 2 min for 16 h. For analysis, nuclear envelope breakdown and anaphase onset time points were determined for cells that completely divided and were detectably expressing GFP.
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