Biotek synergy htx multimode reader

¹H NMR and 2D NMR experiments were performed at 700 and 600 MHz on a Bruker Avance Neo spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA) using CD₃OD as solvent. All chemical shifts were referenced to the residual solvent signal (δ_H 3.31, δ_C 49.0). Through-space ¹H connectivities were evidenced using a ROESY experiment with a mixing time of 200 ms. The HSQC spectra were optimized for ¹J_CH = 145 Hz and the HMBC experiments for ^2,3J_CH = 8.0 Hz. High-resolution MS and LC-MS experiments were recorded on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) combined with a Thermo U3000 HPLC system equipped with a solvent reservoir, inline degasser, binary pump, and refrigerated autosampler. The purification was performed on an HPLC Jasco featuring a quaternary pump, equipped with a Jupiter C18 analytical column (5 μM, 250 mm × 4.6 mm i.d.) and the metabolites were revealed by using a photodiode array detector (PDA).
UV-Vis measurements at 593 nm were performed with the BioTek Synergy HTX Multimode Reader (Agilent Technologies, Santa Clara, CA, USA).

The resazurin assay was used to infer cell metabolic activity in spheroids over time. For each independent experiment, a minimum of 3 spheroids were collected per sampling time and transferred individually to a 96-well plate (non-tissue culture-treated plate, 351172, Falcon, Corning, New York, NY, USA). After that, 90 µL of fresh culture medium was added to each well. A stock solution of 2.2 mM of resazurin (Cayman Chemical Company, Ann Arbor, MI, USA) was prepared in sterilized PBS (1×), and a final concentration of 44 μM per well was obtained by dilution [29 (link)]. A concentration of 10 µM of resazurin was used in preliminary testing, but it proved ineffectual due to low sensitivity in the case of small spheroids. Twelve blank wells were included by adding the same amount of medium and resazurin in the wells but without spheroids. The plates were incubated at 18 °C for 3 h, at constant agitation (~100 rpm) and protected from light. We also incubated for 6 h in addition to 3 h; however, there were no significant variations in the measurements. Fluorescent quantification at 550 nm and 588 nm (excitation and emission lengths, respectively) were read with a microplate Biotek Synergy™ HTX multimode reader (Agilent, Santa Clara, CA, USA) with the software Gen5 (Agilent, Santa Clara, CA, USA). RFU values for each sample were adjusted by blank subtraction and plotted over time.