Synergy mx

Cell viability was determined using the colorimetric Alamar Blue and MTT assays, which measure the reduction of non-fluorescent dyes to the fluorescent metabolites resorufin and formazan, respectively. For both assays, cells were cultivated in 96-well plates. The following day, the cells were treated with cisplatin and/or arzanol for 24 h. For the MTT assay, MTT (Roth #4022) was added to the cells at 0.5 mg/ml concentration and incubated at 37 °C for 1 h. Afterward, the plates were centrifuged at 600 × g and 4 °C for 5 min, and cells were lysed in DMSO for 20 min in the dark. Finally, the absorbance was measured at 570 nm and 650 nm for reference, using a microplate reader (BioTek, Synergy Mx). For the Alamar blue assay, 40 µM resazurin sodium salt (Cayman Chemicals, #14322) was added to the cells and incubated at 37 °C for 3 h. Afterward, the absorbance was measured at 590 nm, using a microplate reader (BioTek, Synergy Mx). The mean of the absorbance of untreated control samples was set as 100%.

For adherent conditions, cells were seeded at 2000 per well in 96-well tissue culture coated plates, flat bottom, in the presence or absence of 80 μl of cisplatin at the concentrations indicated in the figure. After 72 h, 20 μl MTT reagent (Cell titer grow from Promega) were added, incubated for 30 min on a shaker and read on the BioTek Synergy Mx plate reader.
For spheroids, cells were plated at 2000 per well in 96-well ultralow round bottom attachment plates and incubated for 10 days in the presence of 50 μl of cisplatin at the concentrations indicated in the figure. After 10 days, 50 μl of Promega 3D were added, incubated for 30 min on a shaker and read using the BioTek Synergy Mx plate reader. Three independent experiments were performed and IC50 was obtained on prism and averaged.

Changes in free intracellular calcium concentration were measured with the fluorescent indicator Fura-2 AM. BMDMs were loaded for 40 min at 37 °C with 4 μM Fura-2 AM and 0.02% pluronic acid, washed, and fluorescence was recorded by an automatic fluorescence plate reader (Synergy Mx; BioTek, Winooski, VT, USA) for 200 s at 4 s intervals at a wavelength emission couple 340/380 nm, emission 510 nm. ATP was automatically injected into the wells at the designated time points. Intracellular calcium levels were expressed as the ratio of the emission intensities at 340 and 380 nm, and the value was normalised to the fluorescence at time 0 (F/F₀). For Yo-Pro uptake, BMDMs were preincubated for 10 min at 37 °C with 25 μM of punicalagin, following the addition of 2.5 μM Yo-Pro. The images were recorded at 10 s intervals for 60 min before and during injection at 37 °C with ATP, digitonin, or Triton-X100. Yo-Pro bound to DNA fluorescence was measured at 485±9/515±9 nm with bottom excitation/emission in the Synergy Mx plate reader (BioTek). In some experiments Yo-Pro uptake was acquired with a Nikon Eclipse Ti microscope as stated above but using a 20 × objective and time-lapse images were analysed with ImageJ software (US National Institutes of Health) and average of relative fluorescence units recorded from ROI are shown in the results.

Human nondiabetic and diabetic pre-adipocytes were grown to confluency in a 96-well plate and differentiated for 2 days in differentiation media; 1μg plasmid per 1 mL of media for Sort_T, Sort_FL or lipofectamine control was overexpressed for 48 h in differentiation media. Glucose uptake was performed (ProMega #TM467, Madison, WI, USA) by measuring luciferase luminescence. Briefly, cells were serum-starved in Krebs-Ringer Phosphate HEPES buffer (KRPH) for an hour before insulin treatment (100 nM, 10 min). Cells were washed and treated with 1 mM of 2-deoxyglucose 6-phosphate (2DG6P) for 10 min. Cells were washed again in KRPH buffer and lysed. A standard curve was generated, and luminescence was measured on a Biotek Synergy Mx microplate reader (Agilent Technologies, Santa Clara, CA, USA) using a 1 s integration time and 2DG6P quantification of samples were calculated.

3T3-L1 preadipocytes were seeded in a 48-well plate and cultured. After reaching confluence, induced differentiation of the cells was performed as described in Section 4.1. On Day 6, the cells are stained using AdipoRed™ assay reagent (Lonza K.K., Sagamihara, Japan) and fluorescence was measured using a Synergy™ MX microplate reader (Agilent Technologies, Inc., Santa Clara, CA, USA) to detect lipid accumulation.