Spectramax m2e plate reader

3D printed collagen constructs with different concentrations of microfat were cultured individually in a 12 well plate in growth medium composed of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for 2 weeks. Culture medium was replaced every 3 days. Cell metabolic activity was quantified using the AlamarBlue assay by following the manufacturer’s instructions. Briefly, at periodic intervals, microfat-laden collagen constructs were incubated in 500 μl of 10% alamar blue mix (i.e., DMEM + 10% alamar blue solution) for 12 h. Following this, the alamar blue mix from each well was transferred into a 96-well plate in triplicate and the relative fluorescence units were measured at 555 nm excitation and 595 nm emission wavelengths using an M2e Spectramax plate reader (Molecular Devices).

Our assay is outlined
in Figure 1. For each
mutant to be tested, we generated a non-fluorescent version by introducing
an R at position Q183 of GFP.^{26 (link)} For each
comparison to be made, the overnight cultures of fluorescent and non-fluorescent
transformants were normalized and mixed 50/50. The 50/50 mixture was
then used to inoculate 1 mL cultures containing serial dilutions of
cefotaxime and grown overnight in a 96-well deep-well plate. The following
morning, 200 μL of each culture was transferred into black-walled
96-well microtiter plates, and fluorescence (ex: 395 nm, em: 509 nm,
autocutoff: 495 nm) and OD₆₀₀ were measured in tandem on
a Spectramax M2e plate reader (Molecular Devices). The highest cefotaxime
concentration for which there was observed growth (OD₆₀₀ ≥ 0.1) was used for further analysis.

For each logic gate, the transcription
factor plasmid contains a single repressor (BUFFER), single antirepressor
(NOT), repressor pair (AND), antirepressor pair (NOR), or repressor/antirepressor
pair (NIMPLY). The transcription factor and corresponding reporter
plasmids were double-transformed into homemade chemically competent
3.32 E. coli cells (Genotype lacZ13(Oc),
lacI22, LAM–, el4–, relA1, spoT1, and thiE1, Yale CGSC
#5237) and transformants were precultured for 6 hours in LB media
with chloramphenicol (25 μg/mL, VWR Life Sciences) and kanamycin
(35 μg/mL, VWR Life Sciences) antibiotics. Precultures were
then diluted in sextuplicate into glucose (100 mM, Fisher Scientific)
M9 minimal media supplemented with 0.2% (w/v) casamino acids (VWR
Life Sciences), 1 mM thiamine HCl (Alfa Aesar), antibiotics, and respective
inducers, and grown in a flat bottom 96-well microplate (Costar) for
16 hours (37 °C, 300 rpm). Microwell plates were sealed with
Breathe-Easy membranes (Diversified Biotech) to prevent evaporation.
Inducer concentrations used are as follows: isopropyl-β-D-thiogalactoside
(IPTG; 10 mM, reduced to 1 mM for IPTG-fucose gates), D-ribose (10
mM), cellobiose (10 mM), D-fucose (10 mM), fructose (10 mM), and adenine
(1 mM). Optical density (OD₆₀₀) and GFP fluorescence (λ_ex = 485 nm, λ_em = 510 nm) were measured with
a Spectramax M2e plate reader (Molecular Devices).

TcAMT-KO and wild-type epimastigotes at log phase of growth were collected by centrifugation at 1,700 × g for 10 min, washed twice in PBS, and resuspended at a density of 1 × 10⁸/ml in isosmotic buffer (64 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, 150 mM d-mannitol, 5 mM HEPES-Na, pH 7.4). The osmolarity of the buffer was adjusted to 300 ± 5 mosM as verified by an Advanced Instruments 3D3 osmometer (Norwood, MA). Hyposmotic stress was induced by a 1:1 dilution of the cell suspension with sterile deionized water, resulting in a final osmolarity of 150 mosM. Cells were centrifuged at 1,700 × g for 10 min 30 min after dilution and analyzed by qRT-PCR and Western blotting. To monitor the regulatory volume decrease after hyposmotic stress, the cells were subjected to hyposmotic stress, as described above, and absorbance changes at 550 nm were recorded every 6 s for 10 min using a SpectraMax M2e plate reader (Molecular Devices).

PCR experiments were conducted on a PCR instrument and a gel imager (Bio-Rad Laboratories, U.S.A.). Nickel affinity chromatography was conducted on an AKTA purifier equipped with a HisTrap FF column (GE Healthcare, U.S.A.). Scanning diagram of plate colony was conducted on an automatic colony counter (Shineso, China). Fluorescent micrographic imaging of stained bacteria was conducted on a NI−U fluorescent microscope (Nikon, Japan). OD₅₇₀ value was measured on a SpectraMAX M2e plate reader (Molecular Device, U.S.A.).