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Measurement of proteasome inhibition in live cells in Molecular Devices microplate fluorometers

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SpectraMax M5 Multi-Detection Reader (Molecular Devices)

FlexStation Scanning Benchtop Fluorometer and Integrated Fluid Transfer Workstation (Molecular Devices)

Analyst GT Multimode Reader (Molecular Devices)


METHODS
The stock solution of ALLN (a well-characterized proteasome inhibitor) was prepared by dissolving in DMSO to a concentration of 10 mM. The HEK 293 ZsGreen Proteasome Sensor Cells were cultured in DME + 10% FBS + 1% Pen/ Strep/L-glutamine + 200 g/mL of G418. They were seeded overnight in 100 L at a density of 30,000 cells/well in a Costar black-wall /clearbottom 96-well plate. The cells were then treated with ALLN at doses of 0, 0.1, 0.3, 1, 3, 10 and 100 M for 20 hours (N=12/group). The green fluorescence of ZsProsensor-1 in non-treated and treated cells was measured from the bottom in the readers.

Instrument settings
Gemini EM, FlexStation: Ex/Em = 484/510 with a 495 nm emission cutoff filter and PMT set to Auto.

SpectraMax M5: Ex/Em = 484/525 with a 515 nm emission cutoff filter and PMT set to Auto.

Analyst GT: Ex/EM band-pass filters were 48520 and 530-25. A 50% transmission dichroic mirror was used. The sensed volume was focused at 1 mm above the well bottom.


RESULTS
ALLN inhibited proteasome activity in a dose-dependent manner, as expected (Figure 2a-2c). (ALLN is a peptide aldehyde that inhibits the proteasomes chymotrypsin activity and prevents the complex from degrading the ZsProsensor protein, thus allowing it to accumulate and causing an increase in the green fluorescence signal.) The dose-response curves from the three instruments were virtually superimposable if normalized. The EC50 of ALLN was approximately 10 M in all instruments. This value is similar to the reported EC50 value for ALLN inhi
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