Cytation 5 multi mode reader

The sequence of the adenine riboswitch (or mutants) was inserted between enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) genes in the vector pE1K to generate the vector pE1K_add. E. coli DH5α cells carrying the pE1K or pE1K_add were pre-incubated in LB media at 37°C for 8–10 h and then transferred to minimal media and cultured at 25°C until the OD₆₀₀ reached ∼0.3. Culturing of E. coli was continued after the addition of 0, 0.01, 0.1, 0.5 and 1 mM adenine until the OD₆₀₀ was 0.6–0.8. The fluorescence of EGFP (the internal control) and RFP was measured at 510 nm (λ_ex = 480 nm) and 610 nm (λ_ex = 580 nm), respectively, by a Cytation™ 5 multi-mode reader (BioTek, USA). All experiments were performed in triplicate. The expression level of RFP controlled by the adenine riboswitch was determined by: $\text{Expression}\text{level}=\frac{R–R_0}{R_c–R_0}\times \frac{E_c–E_0}{E–E_0}$ whereR and E are the fluorescence of RFP and EGFP from pE1K_add, R_c and E_c are the fluorescence of RFP and EGFP from pE1K, and R₀ and E₀ are the fluorescent background of E. coli DH5α cells at 610 and 510 nm, respectively.

The HEK293 TCF/LEF-reporter recombinant cell line (BPS Bioscience; Cat.# 60,501) was cultured according to the vendor’s recommendations. To avoid detachment of the reporter cells from the culture well surface, L-WRN CM or primary culture medium was carefully added at 1:1 vol/vol to the existing HEK293 medium in the culture well for this assay. Viability in the 1:1 medium mixtures was determined using the CellTiter-Glo® 3D Cell Viability Assay, as described above. Recombinant Wnt3a (R&D systems; Cat.# P27467) was used as a positive control. L-WRN CM batches were tested at a final concentration of 5% (i.e., added 10% L-WRN CM at a 1:1 vol/vol to the HEK293 medium) for Wnt activity assays. Luciferase activity was determined using the ONE-Step™ Luciferase Assay System (BPS Bioscience; Cat.# 60690–2) and luminescence was measured with a Cytation5 Multi-mode Reader (BioTek). Culture wells containing L-WRN CM and ONE-Step™ reagent but without cells were used to determine the average back-ground luminescence. Culture wells containing cells, and HEK293:primary culture medium (1:1) were used to determine baseline reporter activity. Experiments included 3 technical replicates, and 3 biological experiments with distinct passages of cells were performed. Technical replicates from each experiment were averaged prior to statistical analysis.