Redox processes play an important role in cellular physiology and pathology. A particularly powerful tool for the monitoring of cellular redox changes are genetically-encoded biosensors based on redox sensitive green ﬂuorescent protein (roGFP). RoGFPs contain two cysteine residues engineered to be present on the surface of the protein β-barrel, which are capable of forming a disulphide bond. RoGFP can be made to respond to speciﬁc redox species via the genetic fusion of appropriate redox enzymes. For example, fusion of roGFP2 to the thiol peroxidase Orp1 generates an H2O2-sensitive probe.
RoGFP2 exhibits two ﬂuorescent excitation maxima, at 405 nm and 488 nm, when monitoring ﬂuorescence emission at 510 nm. The relative intensities of the two excitation maxima shift in an opposing direction upon reduction or oxidation of the roGFP2 disulphide (Fig. 1). Consequently, by simultaneously monitoring ﬂuorescence emission at the two excitation maxima, it is possible to determine the degree of probe oxidation.
Fluorescence microplate readers would represent an ideal system for roGFP-based high throughput screening, for example, to identify chemical compounds that modulate redox homeostasis. However, microplate reader-based roGFP measurements of cell monolayers require highly sensitive instruments. In this application note, we show that the BMG LABTECH microplate reader enables roGFP2-based measure-ments in mammalian cell monolayers grown in 96-well imaging plates.
Materials & Methods
- BMG LABTECH multimode microplate reader
- Black ﬂ at-bottomed 96-well plates (BD Falcon)
- Hydrogen peroxide (H2O2) (Sigma, H1009)
- Imaging buffer (130 mM NaCl, 5 mM KCl, 10 mM D-glucose, 1 mM MgCl2, 1 mM CaCl2, 20 mM HEPES)
Cells stably expressing the cytosolic H2O2 probe roGFP2-Orp1 were seeded into a 96-well imaging plate (20,000 cells / well). The same number of non-transduced cells were seeded for use as a background control. The cell number was selected so as to obtain 100% conﬂuence on the day of the measurement.
Growth media was removed and the cells were washed twice with PBS, before application of 120 μl of imaging buffer. The response of the probe to an injection of a bolus of H2O2 was followed over time.
|Measurement type:||Fluorescence intensity, Bottom reading|
|Measurement mode:||Plate mode kinetic|
|No. of cycles:||
|No. of flashes:||
|No. 1:||400 520|
|No. 2:||485 520|
Using onboard injectors
Results & Discussion
With the current state feature of the control software, it is possible to follow the reaction progress in real-time. A typical signal curve is shown in Figure 2.
In the sample expressing the roGFP2-Orp1 construct, it can be clearly seen that after H2O2 injection the values measured for 400/520 will increase while the values for 485/520 decrease respectively. No effect can be seen in the no construct or no injection control.
The measurement data was processed to obtain degree of probe oxidation values. In ﬁgure 3 we monitored the response of the roGFP2-Orp1 probe in a monolayer of conﬂuent lung adenocarcinoma cells, following addition of a bolus of H2O2. The sensitivity of the microplate reader makes such measurements easily achievable (Fig. 3).
We next assessed the impact of chemical compounds on cellular redox homeostasis. To this end lung adenocarcinoma cells expressing the cytosolic H2O2 probe roGFP2-Orp1 were treated overnight with different concentrations of the compound of interest. Subsequently, the same cells were challenged with a single bolus of H2O2.
As shown in Figure 4, the compound of interest is found to signiﬁcantly impair cellular recovery from an H2O2 challenge in a concentration-dependent manner. This result indicates that the compound disrupts reducing systems inside the cell and thus may be considered a candidate drug to sensitise cancer cells to chemo- or radiotherapy.
The microplate reader from BMG LABTECH enables monitoring of the ratio-metric ﬂuorescent response of roGFP2-based redox probes in monolayers of mammalian cells.