Figure 3. Qualitative analysis of photobleach protection for Hoechst and GFP. HeLa cells were transduced with CellLight Mitochondria-GFP for 24 hours, then labeled with Hoechst 33342 for 15 minutes. ProLong Live Antifade Reagent was added to Sample A and incubated for 2 hours. Samples incubated with ProLong Live Antifade Reagent (Sample A) retained more signal at all time points, compared to control samples in complete medium alone (Sample B).
Live-Cell Imaging Reagents
Live-cell imaging can be challenging. Not only must staining be optimized for the particular assay readout, spectral compatibility and signal-to-noise level, but live-cell imaging of dynamic processes requires active observation over time.
The choice of medium is particularly important for time-lapse imaging and experiments where cells are exposed to ambient conditions for longer periods. Removing the labeling solution and rinsing with fresh medium will reduce the background for cells you want to continue culturing.
For imaging live cells as a final step (no further culture), a background suppressor can be used to optimize the signal.
Live-Cell Imaging Media
For reliable imaging results with live cells, it is essential that the cells be healthy and maintained as closely as possible to physiological temperature, pH, oxygen tension, and other conditions.
With access to a range of live-cell imaging media and wash buffers you can optimize image clarity, signal-to-noise ratio, and cell viability throughout the imaging period.
- PBS—ideal for cell washing and short-term imaging where prolonged incubation is not required
- Live Cell Imaging Solution—an optically clear physiological solution that helps keep cells healthy for up to 4 hours
- FluoroBrite DMEM—an optically clear solution for long-term imaging and subsequent cell culture
Figure 1. Oxidative stress detection in human osteosarcoma (U2OS) cells.
| Reagent | Cell washing | Short-term imaging | Imaging up to 4 hrs | Long-term imaging |
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| PBS, pH 7.4 |
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| Live Cell Imaging Solution |
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| FluoroBrite DMEM |
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Background Suppression
BackDrop Background Suppressor ReadyProbes Reagent is a set of novel reagents designed to effectively suppress background fluorescence in live-cell imaging samples. BackDrop Background Suppressor is a direct-addition product supplied as a six-pack of ultra-convenient dropper bottles: two vials each of blue, green, and red background suppressors.
If you are observing high background signal or weak fluorescence in the blue, green, or red channels, see how BackDrop Background Suppressor can improve your results by cutting the haze and increasing contrast.
Figure 2. BPAE cells in DMEM, 20% FBS without (–) and with (+) BackDrop Background Suppressor.
Antifade Reagents
Loss of fluorescence through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or of long duration, such as for time-course experiments.
To minimize photobleaching of experimental samples, we have developed a series of antifade reagents for live-cell as well as fixed-cell imaging that have been shown to increase fluorophore photostability in a variety of sample types. Reduced photobleaching means longer tracking for time-course experiments, higher sensitivity, and better quantitation from fluorescencet signals.
For Research Use Only. Not for use in diagnostic procedures.