Synergy neo microplate reader

Steady-state ATPase activity was analyzed using an NADH enzyme-coupled absorbance assay (Montpetit et al., 2012 (link)). Briefly, standard ATPase reactions were prepared with indicated proteins (2 μM for Sub2 or THO, 10 μM for Yra1), in 20 mM HEPES (pH 7.0), 100 mM NaCl, 2 mM MgCl₂, 1 mM TCEP, 10 μM poly(U) 20-mer RNA (unless otherwise stated), 1 mM ATP, 2 mM phosphoenolpyruvate, 0.2 mM NADH, and 1% (vol/vol) pyruvate kinase/lactate dehydrogenase (Sigma). UV absorbance at 340 nm was monitored by a BioTek Synergy NEO Microplate Reader at 37°C. Reaction rates were calculated from the slopes of the linear phase showing the decrease in NADH absorbance as a function of time.

For assays in 96 well plates 2500 cells were plated per well. After 24 hours, cells were treated with small molecule inhibitors and incubated for 72 hours at 37°C. Subsequently the cells were lysed and the ATP content was measured as an indicator of metabolically active cells using the CellTiter-Glo assay (Promega). IC₅₀ values were calculated using the GraphPad software. For assays in six well plates, 100,000 cells were plated per well. After 24 hours, cells were treated with small molecule inhibitors and incubated for varying time points. Cells were trypsinized and a suspension was made in 5 ml of phosphate buffered saline. 30 µl of this suspension was mixed with 30 µl of CellTiter-Glo reagent followed by a 10-minute incubation at room temperature. Luminescence was measured using EnVision 2104 Multilabel Reader (PerkinElmer) and BioTek Synergy Neo Microplate Reader.

DCFH-DA (#S0033, Beyotime, Shanghai, China) was used to detect intracellular ROS levels. Human NPCs were plated in Matrigel-coated 96-well black plates (#3094,Corning, NY, USA) at 2000 cells per well and cultured in medium consisting of 1:1 DMEM/F12: Neurobasal, NEAA, Glutamax, N2 and B27 supplement, 1 ng/mL bFGF. The cells were treated with 10 μM Aβ_1-42 for 24 h and then stained with 10 μM DCFH-DA and 3 μg/mL Hoechst (#C1022, Beyotime, Shanghai, China) for 30 min at 37 °C in a humidified incubator with 5% CO₂. The cells were washed twice with PBS and observed under a laser-scanning confocal microscope (Operetta, Perkin Eimer, MA, USA). Alternatively, the fluorescence was measured by a BioTek SynergyNEO microplate reader (Bio-Tek, Williston, VT, USA) at a 488 nm excitation wavelength and a 525 nm emission wavelength.

To assess mitochondrial membrane potential, the lipophilic cationic JC-1 dye was used as part of the JC-1 mitochondrial membrane potential assay kit (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions. Briefly, 24 h following transient transfection of lentiCRISPR-gRNA for telomere or mock plasmids, SH-SY5Y or HEK-293T cells were plated in wells of a 96-well plate at a density of 5 × 10⁵ cells/well. After 48 h of cell culture in the complete medium, cells were stained with JC-1 (10 uL of the JC-1 staining solution per 100 uL of culture medium) for 20 min in the cell culture incubator and washed twice with assay buffer. JC-1 fluorescence were measured by using a SYNERGYneo microplate reader (Bio-Tek, Winooski, VT, USA) with excitation and emission wavelengths of 535 and 595 nm, respectively, emission for healthy mitochondria and 485 and 535 nm, respectively, for unhealthy mitochondria. The relative healthiness of mitochondria membrane potential was expressed as ratio of fluorescence intensities obtained at two different excitation/emission settings.

Polyethyleneimine (PEI, branched, M_w 25000), 3‐(2‐pyridyldithio)propionic acid N‐hydroxysuccinimide ester (SPDP) were obtained from Sigma‐Aldrich. Methoxy poly(ethyleneglycol) propionic acid N‐hydroxysuccinimide (Methoxy‐PEG‐NHS, M_w 5000) was purchased from Nanocs. CpG1826 was obtained from Integrated DNA Technology. Antigen peptides used in this study were synthesized by Genemed Synthesis, which include epitopes of ovalbumin peptide SIINFEKL and CSSSIINFEKL, neo‐epitopes of MC38 colon carcinoma ASMTNMELM (Adpgk) and CSSASMTNMELM (CSS‐Adpgk), neo‐epitopes of B16F10 melanoma LCPGNKYEM (M27), VDWENVSPELNSTDQ (M30), and CSSVDWENVSPELNSTDQ (CSS‐M30). All other reagents were received from Fisher scientific unless otherwise indicated. UV–Vis absorption and fluorescence spectra were obtained using BioTek synergy neo microplate reader. GPC and HPLC were performed using Shimadzu HPLC system equipped with TSKgel G3000SWxl column (Tosoh Bioscience LLC) and Jupiter® C18 LC Column (Phenomenex), respectively. TEM images were acquired using JEOL 1400‐plus. Hydrodynamic size and zeta potential were measured using Zetasizer Nano ZSP (Malvern Panalytical). Flow cytometry was performed using ZE5 Cell Analyzer (Bio‐Rad) and the data were analyzed using FlowJo 10.5 software.