Synergy 2 biotek microplate reader

Black Nunc Maxisorp plates (Nalgene Nunc International, Rochester, NY, USA) were coated overnight with 5 μg/mL 4LCA (Figure 1A–D) or rabbit anti-BoNT/A HC50 antiserum in phosphate buffer saline (PBS) followed by three washes in PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 2% non-fat milk in PBST for 1 h at 37 °C, followed by three PBST washes, and then serial dilutions of RI-BoNT/A in plasma (50 μL each), incubated for 1 h at 37 °C, followed by a 3× PBST wash. Either PBS or 5 μg/mL of the biotinylated mAb (6A or 3B3), diluted in PBS, were added to the samples, incubated for 1 h at 37 °C, and washed 3× in PBST. Streptavidin-poly-horseradish peroxidase conjugate (Thermo Fisher Scientific, Waltham, MA, USA) was added (1:2000), incubated for 1 h at 37 °C, and washed 3× with PBST. Super Signal ELISA Femto Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used for detection and relative luminescence values were measured using Biotek Synergy II Microplate reader (BioTek Instruments, Winooski, VT, USA). Excel was used to process the data. MAbs were biotinylated using the EZ-Link™ Hydrazide-Biotin kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Protein concentration was measured using the NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA).

Luciferase reporter assay was performed as previously described 20 (link), 21 (link), 23 (link), 24 (link). The ARE-luciferase reporter plasmids were transfected into the indicated RTECs using Lipofectamine 3000 reagent (Invitrogen). Each of transfection was included the same amount of pRL-TK Renilla plasmid, which was employed to standardize transfection efficiency. At 48 h after transfection, the luciferase activities in cell lysates were determined with the luciferase assay system (Promega) using a BioTek Synergy2 Microplate reader (Bio-Tek) at wavelengths of 560 and 465 nm according to the manufacturer's guidelines.

For the cytotoxicity evaluation,
1000 cells/well of U251 or T98G
lines were seeded in 96-well plates and left to adhere for 24 h. After,
the cells were treated for 72 h with 100 μL of free TMZ (10
nM to 2.0 × 10⁶ nM), free BTZ (1.0 × 10^–4 to 2.0 × 10³ nM), or free TMZ+BTZ. A constant TMZ:BTZ
ratio (1:0.8) was used for the combined therapy, with concentrations
ranging between 1.0 × 10^–5 and 5.0 × 10² for TMZ, 8.0 × 10^–6 and 4.0 ×
10² nM for BTZ. The studied TMZ:BTZ ratio was chosen based
on both drugs’ EE (%) in the developed NPs. Untreated cells
were used as the negative control. After treatment, the cells were
fixed with trichloroacetic acid (TCA) 10% (w/v) at 4 °C for 1
h, then washed with ultrapure water and dried at room temperature.
Then, cells were stained with SRB for 20 min. The samples were washed
twice with 1% (v/v) acetic acid and air-dried to remove the unbound
dye. The protein-bound dye was solubilized by a Tris buffer solution.
The cell protein quantification was measured by UV–vis absorbance
(BioTek Synergy 2 microplate reader, BioTek, U.K.) at 560 nm. Cell
survival inhibition as a function of drug concentration was then plotted
using GraphPad 9.3.1 software (GraphPad Software Inc., USA).

Glutathione levels were measured using a glutathione assay kit (Cayman Chemical) according to the manufacturer's instructions. Briefly, cells were re-suspended in MES buffer (0.4 M 2-ethanesulphonic acid, 0.1 M phosphate, 2 mM EDTA, pH 6.0) and lysed by sonification. After centrifugation at 10,000g for 15 min, 1.25 M metaphosphoric acid was added to the supernatant for precipitation of proteins, followed by centrifugation at 15,000g for 10 min. The supernatant was transferred into a 96-well plate and incubated with the assay cocktail containing MES-buffer, co-factor and enzyme mixture, and Ellman's reagent for 30 min. Absorbance was measured at 405 nm with a BioTek Synergy 2 microplate reader (BioTek).