Spectramax multiwell plate reader

E. coli BL21DE3 cells transformed with pCWori-Dendra2 were
cultured in LB
medium to an OD₆₀₀ of 0.8. Cells were then resuspended
in M63 minimal media and induced with 0.5 mM IPTG for 3 h at 37 °C
with 180 rpm shaking. Photoconversion of Dendra2 was carried out with
a 405 nm LED flood array (Loctite) with a total light exposure time
of 2 min. Cells were then plated in 96 well plates at 6 × 10⁷ cells per well. Green and red emission was measured directly
after photoconversion using a SpectraMax Multiwell Plate Reader (Molecular
Devices) for a baseline evaluation of Dendra2 protein (t = 0 h). For green emission, an excitation wavelength of 491 nm and
emission wavelength of 538 nm was used; for red emission, the excitation
wavelength was 544 nm and emission wavelength was 590 nm. Compounds
were then dosed from 0 to 300 μM, and compound 1 was activated with light as described above. The cells were incubated
for 16 h before the green and red emission was measured again for
an evaluation of protein synthesis with compound treatment (t = 16 h).
The average fluorescence ratio of green/red
at t = 0 and 16 h was calculated, the values were
normalized, and the data fitted to a sigmoidal dose response.

E. coli BL21(DE3) cells transformed with pCWori-Dendra2
plasmid were plated
in M63 minimal medium at 4 × 10⁶ cells per well in
96 well flat bottom transparent tissue culture treated plates (Greiner
Bio One). Compounds were dosed from 0–300 μM, followed
by 3 min of light irradiation (7 J/cm² blue light (>400
nm)). The cells were then incubated for 16 h with the compounds, and
cell growth was determined by measurement of the optical density at
600 nm using a SpectraMax Multiwell Plate Reader (Molecular Devices).
HL60 cells were plated in Opti-MEM supplemented with 1% FBS and
50 U/mL of penicillin/streptomycin at 30 000 cells per well
in 96 well flat bottom transparent tissue culture treated plates (Greiner
Bio One). Compounds were dosed from 0–300 μM and incubated
for 16 h, followed by light irradiation with 7 J/cm² blue
light (>400 nm) in 30 s pulses for a total light exposure of 3
min.
The cells were then incubated for 72 h, and cell viability was determined
by conversion of resazurin to resorufin. Dark controls were run in
parallel. The emission of resorufin was measured on a SpectraFluor
Plus Plate Reader (Tecan).
The data were normalized to the untreated
control and fitted to
a sigmoidal dose response model using Prism 6.02 to determine IC₅₀ values. Minimal inhibitory concentration (MIC) was fitted
to the model published by Lambert et al. using Prism 6.02.^{32 (link)}