Lab Techniques>Fluorescence Microscopy Techniques Practice Test

Researchers examined ligand-dependent trafficking of a transcription factor by expressing GFP-tagged p65 (NF-κB subunit) in HeLa cells and counterstaining nuclei with Hoechst. Live-cell confocal imaging captured the same cells before and 10 minutes after TNFα treatment. At baseline, green fluorescence was predominantly cytoplasmic with minimal overlap with the blue nuclear signal. After TNFα, green fluorescence concentrated within the Hoechst-positive region, and cytoplasmic green intensity decreased proportionally. Integrated GFP intensity per cell (whole-cell ROI) remained unchanged across time points, and photobleaching was minimized by identical low laser power for paired images. Immunoblotting of total p65 within 10 minutes of TNFα showed no change in protein levels. Cell morphology remained intact without membrane blebbing or nuclear fragmentation during the imaging interval. The investigators conclude that the ligand regulates subcellular localization rather than synthesis or degradation over this acute time window.

To test mitochondrial targeting, COS-7 cells expressed a YFP-tagged matrix presequence and were stained with MitoTracker Red CMXRos, a potential-dependent dye. Under control conditions at 37°C, YFP appeared as puncta distributed throughout the cytoplasm with strong overlap with MitoTracker (Pearson r ≈ 0.82). Upon treatment with the protonophore CCCP for 10 minutes, MitoTracker fluorescence decreased and YFP redistributed diffusely throughout the cytosol with markedly reduced overlap (r ≈ 0.18), despite unchanged total cellular YFP intensity. A temperature shift to 4°C produced a similar loss of colocalization, which was reversible upon return to 37°C. After washout of CCCP, both MitoTracker signal and YFP–mitochondria colocalization recovered. Immunoblotting over this timeframe showed no change in YFP-tagged protein abundance. These observations were replicated in three independent experiments with consistent quantification of colocalization coefficients.