Synergy h4 hybrid microplate reader

Inhibition of cancer cell growth by Ara-C/DS was determined by sulforhodamine B (SRB) colorimetric assay according to previously published protocols [35 (link)]. Briefly, cells were plated at 5 × 10⁴ cells/mL (100 µL in a 96-well plate), rested overnight, and treated with titrated doses of Ara-C or Ara-C/DS. After 48 h of incubation, cells were fixed in 10% trichloroacetic acid (TCA, Fisher Scientific, Fair Lawn, NJ, USA) and stained with 0.4% SRB dye (Alfa Aesar, Heysham, UK). After washing and drying, 200 μL of 10 mM Tris solution (Fisher Scientific, Fair Lawn NJ, USA) were added to the plates and the absorbance was read at 510 nm with the Synergy H4 Hybrid Microplate Reader (Agilent, Santa Clara CA, USA). The % growth was determined in relation to a 0% cell growth (untreated cells fixed at 0 h) and a 100% cell growth (untreated cells fixed at 48 h).

The MIN6 cells were seeded in a 12-well plate to 80–90% confluency. Cells were starved for 1 h in 1 mL KRB (114 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES, 2.5 mM CaCl2, 25.5 mM NaHCO3 and 0.2% BSA) containing 1 mM glucose. They were then cultured with freshly prepared KRB with 1 or 25 mM glucose for another hour. In glucose response curve experiment, cells were cultured in freshly prepared 1 mM, 5.6 mM, 11 mM and 25 mM KRB for another hour after starvation. The conditioned buffer was collected and centrifuge at 2,000 rpm for 5 min to obtain supernatants. The cells were lysed with RIPA buffer (R0278, Sigma) followed by centrifugation to obtain supernatants. The supernatants were then measured for NanoLuc using Promega Nano-Glo® Luciferase Assay System (Promega) or for insulin using a mouse insulin ELISA kit (Mercodia), and DNA using AccuBlue® dsDNA Quantitation kit (Biotium) following manufacturer's instruction. The reactions were read using a BioTek Synergy™ H4 Hybrid Microplate Reader. NanoLuc and Insulin content was normalized to total DNA content (AccuBlue® dsDNA Quantitation, Biotium) in each sample. At least three independently generated sets of samples were analyzed under each condition.

For FPGS enzymatic activity measurement, FPGS cDNAs were cloned into a pFastBac expression vector, and mutations were generated with the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent). Wildtype and R419W, R141H, V136F and K215_V218delinsSP viral FPGS plasmids were transduced into Sf9 insect cells and the indicated FPGS proteins were purified. FPGS proteins were quantified by comparison with the gradient BSA running on the same PAGE gel. FPGS proteins (2.5/5 ng) were applied to 10 µl reaction buffer :(20 mM Tris (pH8.85), 20 mM MgCl₂, 20 mM KCl, 10 mM NaHCO₃, 100 mM 2-mercaptoethanol, 0.5 mg/ml BSA, 5 mM glutamic acid), 5 µl MTXpg1-6(0.4 µM), 2.5 µl ATP(60 µM) and 10 µl phosphate sensor(2x), incubated at 37 °C for 5 ~ 10 min. Fluorescence was read on BioTek Synergy H4 Hybrid Microplate Reader at 450 nM.
The enzymatic activity of FPGS mutant proteins were tested in triplicate and normalized to the FPGS wild type protein, and the results were plotted using Graph Prism (version 7.05). All experiments were conducted independently. The data are expressed as the means ± the standard error (SEM). Group means were compared with Student’s t test for two groups. P values < 0.05 were considered statistically significant.

The purification step involved removal of the non-encapsulated dye from liposomes and EMVs by gel filtration using a PD-10 column (GE Healthcare, UK) equilibrated with 1× PBS. Briefly, 1 mL of the extruded sample was inserted into the column, and seven fractions (1 mL each) were collected. The presence of liposomes/EMVs was proved via particle size and absorbance measurements of each fraction. Only in fractions 3 and 4, liposomes with a size of around 115 nm and a considerable count rate were detected, which was further supported with absorbance measurements using 96-well transparent F-bottom microplates and a Synergy H4 Hybrid microplate reader (BioTek, Winooski, VT, USA). A 2× dilution factor was considered when calculating the encapsulation efficiency (EE%). The same procedure was applied for EMVs. Fractions 4 and 5 were collected, having a considerable count rate and a particle size of 220–230 nm on average.