ProLong Live Antifade Reagent suppresses photobleaching and preserves the signals of your fluorescently labeled target molecules for longer-term imaging and analysis, even with high-intensity illumination. After 120 exposures using a standard time-lapse imaging protocol, samples treated with ProLong Live reagent are more than 20% brighter than untreated cells, allowing you more time to collect data from your imaging experiment.
Add ProLong Live Antifade Reagent to your live-cell imaging medium to reduce photobleaching and image your cells for longer. It is common practice for researchers to track cellular behavior until the signal intensity degrades to 50% of the starting level. With ProLong Live Antifade Reagent, you can image up to 20% longer before reaching that 50% signal level, to see more of the cellular processes you are following.
With ProLong Live Antifade Reagent you can preserve the signals of fluorescent proteins, functional probes like MitoTracker dyes, and counterstains such as Hoechst 33342 (Figure 1) for extended imaging protocols and more accurate measurements.
Figure 1. 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 B and incubated for 2 hours. Samples incubated with ProLong Live Antifade Reagent (A) retained more signal at all time points, compared to control samples in complete medium alone (B).
ProLong Live Antifade Reagent preserves the signal in live cells for a range of fluorophore types (Figure 2).
Figure 2. Effective photobleaching protection with multiple fluorophores. HeLa or U2OS cells expressing fluorescent proteins (CellLight MitoGFP and MitoRFP) or treated with fluorescent dyes (MitoTracker Green, Red, or Deep Red or Hoechst 33342 dye) were treated with ProLong Live reagent and scanned using a high-content analysis instrument in confocal mode. The degree of photobleaching protection is expressed as the fraction of initial fluorescence intensity remaining following repeat exposures. EC50 (half-maximal fluorescence signal) is indicated for each graph, and the overall signal protection offered by ProLong® Live reagent (compared to untreated samples) is calculated based on the scan number where treated and untreated samples reach the EC50 value. The addition of ProLong Live reagent permitted (A) 50% more captures with CellLight MitoGFP (EmGFP); (B) 100% more captures with CellLight MitoRFP (TagRFP); (C) 120% more captures with MitoTracker Green; (D) 67% more captures with MitoTracker Red; (E) 85% more captures with MitoTracker Deep Red; (F) More captures (amount not calculated) with Hoechst 33342.
Incubation with ProLong Live Antifade Reagent, used according to the protocol, has minimal effect on cell viability or proliferation (Figure 3).
Figure 3. Cell viability and proliferation are not affected by ProLong Live reagent treatment. HeLa cells were plated at a concentration of 1,000 cells/well in a 96-well plate and treated with a working concentration of ProLong Live Antifade Reagent for the period indicated. Cell viability was detected using A.LIVE/DEAD Red dye, B.CyQUANT Direct reagent, and C.PrestoBlue reagent. Cell proliferation was detected with D.Click-iT Plus EdU reagent. Error bars = standard deviation.
For Research Use Only. Not for use in diagnostic procedures.