ProLong Live Antifade Reagent Protects Fluorescent Proteins and Dyes In Live-Cell Imaging

At long last, protection from photobleaching for live cells

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One of the key pain points when labeling cells and tissues with fluorescence is photobleaching, the degradation of fluorescent signals as samples are exposed to light. Photobleaching is a complex photodynamic process whereby a photoexcited fluorophore interacts with molecular oxygen, resulting in the destruction of the fluorophore and the production of highly reactive singlet oxygen (¹O₂) that can further degrade neighboring dye molecules. This loss of signal is particularly problematic when attempting to collect time-course data, image rare targets with low signal-to-noise ratios, or quantitatively compare fluorescently labeled samples. In this article we describe the recently introduced ProLong™ Live Antifade Reagent, which helps protect fluorescent proteins and dyes from photobleaching in live cells.

How to protect live cells from photobleaching

Researchers using fixed-cell systems have long had the luxury of a number of commercial antifade mounting media to choose from, including the recently released ProLong™ Diamond and SlowFade™ Diamond Antifade Mountants. These fixed-cell mountants contain antioxidants and free radical scavengers that greatly reduce the rate of photobleaching; these formulations, however, are not intended for use with live-cell systems. Factors such as pH, osmolarity, and nutrient content in fixed-cell mountants are not balanced for live cells, and the antifade components themselves may be cytotoxic.

Consequently, when imaging live cells, researchers have relied on their own in-lab antifade formulations, such as Trolox™ antioxidant [1,2] or ascorbic acid added to cell environments. Unfortunately, the protection from photobleaching provided by these additions (at concentrations that do not perturb cell function) is not significant for most fluorescent dyes and virtually nonexistent for fluorescent proteins. Without an effective live-cell antifade option, the best course of action for live-cell imaging has been to minimize light exposure as much as possible [3,4] by limiting exposure time, light intensity, and frequency of sampling [3], and to use only the most photostable dyes, which severely reduces fluorophore choices.

ProLong Live Antifade Reagent protects live cells...

After a comprehensive review of many different formulations, we offer ProLong Live Antifade Reagent. Based on Oxyrase™ technology [5], ProLong Live reagent contains enzymes from the plasma membrane of naturally occurring E. coli. These enzymes metabolize environmental components that exacerbate photobleaching, and they are not cell permeant so intracellular functions are minimally affected.

ProLong Live Antifade Reagent is diluted into cell medium or a suitable imaging buffer, such as FluoroBrite™ DMEM, and then added directly to cells for a 15- to 120-minute incubation. After incubation, imaging can be performed for up to 24 hours with continuous protection from photobleaching. ProLong Live Antifade Reagent has been validated to provide protection for a range of live-cell–compatible organic dyes, including Hoechst™ 33342, MitoTracker™, LysoTracker™, and CellTracker™ dyes; Figure 1 shows representative photobleaching curves. Furthermore, ProLong Live Antifade Reagent can be used for dyes across the spectrum without any initial quenching or increased background fluorescence.

Importantly, ProLong Live Antifade Reagent also protects fluorescent proteins from photobleaching. Compared with cells in medium only, use of ProLong Live Antifade Reagent with emGFP or TagRFP allows you to acquire many more images before the sample fades to half of the initial fluorescence intensity (Figures 1B and 1C). The images in Figure 2 show this protection from photobleaching over time for HeLa cells labeled with both emGFP and Hoechst 33342 dye.

Figure 1. Quantitative analysis of protection from photobleaching by ProLong Live Antifade Reagent for key dyes and fluorescent proteins. HeLa or U2OS cells were stained with (A)Hoechst™ 33342, (B)CellLight™ Mitochondria-RFP, (C)CellLight™ Mitochondria-GFP, or (D)MitoTracker™ Green FM reagents. Cells were incubated for 2 hr in the dark either in complete medium (control) or in complete medium containing ProLong™ Live Antifade Reagent. After incubation, cells were imaged every 15 sec using optimal but consistent excitation/emission imaging conditions with the Thermo Scientific™ ArrayScan™ VTI HCS Reader.

Figure 2. Qualitative assessment of protection from photobleaching by ProLong Live Antifade Reagent for Hoechst 33342 and GFP. HeLa cells were transduced with CellLight™ Mitochondria-GFP for 24 hr, then stained with Hoechst™ 33342 for 15 min. ProLong™ Live Antifade Reagent was added to one sample, while the second sample remained in complete medium. After a 2 hr incubation, samples were imaged every 15 sec for a total of 240 total images, using the same exposure conditions for each image and sample. The sample incubated with (A) ProLong Live Antifade Reagent retained more signal at all time points, when compared to the sample in (B) medium alone.

...Without affecting cell viability or proliferation

ProLong Live Antifade Reagent has been rigorously tested and shows little to no measurable effect on cell vitality, proliferation, or incidence of apoptosis over 48 hours (Figure 3). Using Dead Red Stain (a component of the LIVE/DEAD™ Cell Imaging Kit, Figure 3A), which only enters and fluoresces in dead cells, we showed that after 48 hours there was no significant decrease in the percentage of live HeLa cells in the presence of ProLong Live reagent. Likewise, cells stained with PrestoBlue™ Reagent (Figure 3B), which is converted to fluorescent resorufin by cellular metabolism, showed no significant decrease in fluorescence after 48 hours in the presence of ProLong Live reagent, indicating no change in cell vitality. And for cell proliferation, the cells’ ability to divide normally in the presence of ProLong Live reagent for up to 48 hours was demonstrated with the Click-iT™ Plus EdU Assay (Figure 3C), which measures DNA synthesis, and the CyQUANT™ Direct Assay (data not shown), which measures total DNA. Finally, using CellEvent™ Caspase-3/7 Green Detection Reagent (Figure 3D), we have shown that ProLong Live Antifade Reagent does not induce apoptosis over a 48-hour time period. ProLong Live Antifade Reagent can help you maintain longer imaging times for scans or time-lapse experiments, and enable you to detect low-abundance targets without sacrificing cell health.

Figure 3. Assessment of cell viability and proliferation in the presence of ProLong Live Antifade Reagent. HeLa cells were plated at a concentration of 1,000 cells/well in a 96-well plate using Gibco™ MEM (Cat. No. 11095-080) with 10% FBS (Cat. No. 16000-036). A working concentration of ProLong™ Live Antifade Reagent (Cat. No. P36974) was added to 8 replicate wells, every 6 hr, for up to 48 hr, while 8 wells without antifade reagent served as a control. Cell viability was detected using (A) Dead Red Stain (a component of the LIVE/DEAD™ Cell Imaging Kit (488/570)) and (B)PrestoBlue™ Cell Viability Reagent, (C) cell proliferation was detected with Click-iT™ Plus EdU Reagent, and (D) apoptosis was detected with CellEvent™ Caspase-3/7 Green Detection Reagent using the ArrayScan™ VTI HCS Reader. These data demonstrate that the presence of ProLong Live Antifade Reagent has no statistically significant effect on cell vitality, proliferation, or incidence of apoptosis over a 48 hr time period. Error bars = standard deviation.

References

1. Rasnik I, McKinney SA, Ha T (2006) Nat Methods 3:891–893.
2. Cordes T, Vogelsang J, Tinnefeld P (2009) J Am Chem Soc 131:5018–5019.
3. Frigault MM, Lacoste J, Swift JL, et al. (2009) J Cell Sci 122:753–367.
4. Waters, JC (2013) Methods Cell Biol 114:125–150.
5. Thurston M, Maida D, Gannon C (2000) Lab Med 31:509–512.