2030 multilabel reader victor x5

Cells were aliquoted into 96-well plates at a density of 2000–5000 cells/well and incubated overnight at 37 °C. The medium was then removed and replaced with medium containing the indicated amount of the HIF-1α-stabilizing drug. After incubation with the drug for 24 h, the cells were washed with PBS and incubated in drug-free fresh media for another 72 h. The cells were then washed with PBS, and cell viability was assayed by measuring ATP levels using a luciferase-based CellTiter-Glo kit (Promega, Madison, WI, USA) in a VICTOR X5 2030 Multilabel Reader (PerkinElmer, Waltham, MA, USA). When cells were treated with GCV in addition to the HIF-1α-stabilizing drug, they were seeded at a density of 1000–3000 cells/well and incubated overnight at 37 °C. The cells were then incubated for 24 h with the indicated amount of DFO or DFX on days 1 and 5 of the experiment and with 20 μg/mL of GCV on the other days prior to assaying for cell viability. Luciferase activity levels for drug-treated cells were normalized to levels obtained with cells incubated in parallel in the absence of drug. p-values comparing cell viability of EBV-positive to EBV-negative cells were calculated with a Student’s t-test.

The commercially available CellTiter-Glo^® luminescent cell viability assay kit (Promega) was used with the following modifications: To quantify intra-cellular ATP, 25 µl CellTiter-Glo was added to the remaining cells in the 96-well plate and 20 µl CellTiter-Glo was added to the remaining microtissues in the capillaries. The cells and microtissues were then incubated at room temperature for 30 min. The detection of the luminescence signal was performed in a white microtiter plate using a VICTOR X5 2030 Multilabel Reader (Perkin Elmer, MA, USA).

The siRNA-loaded SSNs with or without PEG were formulated using 40 mM of SrCl₂, 10 mM of Na₂SO₃, and 1 µL of 1 M biotin PEG in 50 µL of aqueous solution. After incubation at 37 ◦C for 30 min, SSNs/PEG-SSNs/siRNA complexes were subsequently suspended into 950 µL DMEM media of different pHs (7.5, 7.0, 6.5, 6.0, 5.5, and 5.0). Afterward, the particle suspensions were centrifuged at 6000× g rpm for 20 min at 4 °C with a refrigerated bench-top microcentrifuge. The supernatant was discarded and the pellet was mixed with 10 mM EDTA in PBS to dissolve the particles and release the bound siRNAs, prior to fluorescence intensity measurement with an excitation wavelength of 490 nm and an emission wavelength of 535 nm using PerkinElmer 2030 manager software attached with 2030 multilabel reader victor X5 (PerkinElmer, Waltham, MA, USA). The concentrations of AF-488 siRNA present in the suspension were calculated from the fluorescence intensity, using the standard curve. Data were represented as the % of siRNA release from NPs. The % siRNA release profile was calculated using the following formula:
where “[Y] NP bound siRNA at different pH” is the fluorescence intensity of siRNA bound with NPs at pH 7.0, 6.5, 6.0, 5.5, and 5.0 and “[X] NP bound siRNA at pH 7.5” is the fluorescence intensity of AF-488 siRNA bound with NPs at pH 7.5. Data were represented as mean ± SD for triplicates.