Microplate reader

Cells cultured in SC medium for 6 h and 12 h were collected and washed twice with sterile water before cell lysis. After the addition of RIPA lysis buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 3 mM EDTA) and glass beads, the mixture was violently shaken and placed in an ice-bath. This was repeated about 10 times for 2 min each. The mixture was centrifuged at 12,000 rpm for 25 min; the supernatant was used for TG measurements. TG measurements were based on the method detailed in the TG measurement kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In detail, 1 mL of working fluid was mixed with 10 μL of cell lysate and incubated at 37 °C for 30 min. The absorption of the mixture at 500 nm was then detected with a microplate reader (PerkinElmer LLC, Boston, MA, USA). The final TG concentration is normalized by the protein concentration, which was determined using a BCA protein assay kit (Solarbio, Beijing, China).

Cell viability was determined with the thiazolyl blue tetrazolium bromide (MTT) assay as previously described [22 (link)]. Briefly, 1 × 10⁴ cells were seeded in the wells of a 96-well microplate and cultured with α-syn, p-α-syn, sonicated OW, OP, PFF, and pPFF for 1, 3, and 6 h. Afterward, the medium was replaced with MTT (M2128; Sigma-Aldrich Corporation) at a final concentration of 0.5 mg/mL and incubation was continued for 4 h. Then, the medium was discarded and replaced with 100 μL of dimethyl sulfoxide to dissolve the formazan crystals. Absorbance was measured at 490 nm with a microplate reader (PerkinElmer, Inc., Waltham, MA, USA). Three independent experiments were performed.

The proliferation ability was analysed using the Cell Counting Kit-8 (CCK-8 Kit, Dojindo Laboratories, Japan) assay. Glioma cells (5000 cells/well) were seeded into 96-well plates (Corning, USA) at 100 μl and incubated overnight. Then, the medium was changed to serum-free DMEM medium, and TMZ was added as scheduled. Ten microlitres of CCK-8 solution was added at various time points, and the absorbance value at 450-nm wavelength was detected by a microplate reader (PerkinElmer, USA) after 2 h of incubation. The migration ability was investigated using a wound-healing assay. Glioma cells were seeded into 6-well plates and incubated until they reached 90–100% confluence. A 10-μl pipette tip was used to carefully make cross lines, and the debris was washed away with PBS. Different medium with 200 μM TMZ was added. The areas of the scratch wounds were imaged with an Olympus microscope at 0 and 24 h and analysed using ImageJ software (NIH, USA). The apoptosis ability was analysed by flow cytometry (FCM). Glioma cells were treated with TMZ for various times and then collected. Glioma cells were washed and processed to FCM by using an annexin V-APC/7-AAD or annexin V-FITC/PI apoptosis assay kit (Nanjing KeyGEN, China) in accordance with the manufacturer’s instructions.

MTT assay was used to measure the cell viability in strict accordance with the manufacturer's instruction. In brief, cells administered with LPS for 6 h with or without galectin‐1 were seeded at the density of 1 × 10⁴ cells/well in 96‐well plates and cultured for 24 h at 37°C. MTT (Sigma‐Aldrich, M2128) was added to the cells at a final concentration of 0.5 mg/ml, and the cells were incubated at 37℃ for another 4 h. Finally, cells were washed with PBS for three times and formazan crystals were dissolved in 100 ml DMSO. The absorbance was read at 490 nM using a microplate reader (PerkinElmer, USA).

The cytotoxic activity was evaluated by the MTT method. The herbal extracts and selected standards were dissolved in DMSO at the concentrations of 400 mg/mL and 50 mM, respectively. Then, the solutions were diluted in culture medium to obtain gradient concentrations. Celastrol (10 μM) served as the positive control. A2780 cells (7 × 10⁴ cells/mL) were plated into 96-well plates at 100 μL/well. After 18 h of incubation, the medium was replaced by different concentrations of extracts or compounds. After incubation for 48 h, 100 μL of MTT (1 mg/mL) was added into each well and incubated for 4 h. Then the culture medium was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The absorbance of the resulting solution was recorded at 570 nm using a microplate reader (Perkin Elmer, Singapore City, Singapore). Three independent experiments were performed in triplicate.

Cell viability was determined by using a CCK-8 assay kit in 96-well plates. 10 μl of CCK-8 reagent was added to each well and then incubated for 3 h in darkness. The absorbance was detected at 450 nm using a Perkin Elmer Microplate Reader. The mean optical density (OD) of each group was used to calculate the percent of cell viability with the following formula: cell viability = treatment group OD/control group OD × 100%.

The metabolic activity of hDFSCs was determined using Alamar Blue Assay. hDFSCs (10⁴ cells) were seeded onto each well of 48 well plate, followed by 48 h incubation (37 °C and 5% CO₂) in normal complete medium containing different amount of NS (NC − F and NC + F) (0 µg.mL⁻¹ to 100 µg.mL⁻¹). After incubation, the medium was removed and cells were washed with 1X PBS. These nanosilicates incubated hDFSCs were further cultured in three different media such as NCM, OCM, and OIM for 14 d. The assay was performed on the day 1, 7, and 14, according to the manufacturers protocol. In brief, after predetermined duration, the media was removed and the cells were washed twice with PBS. Then the PBS was replaced with 10% (v/v) of alamar blue reagent in complete medium and incubated at 37 C, 5% CO₂ for 3 h. After incubation, the supernatant of the cultures was aliquoted into 96-well plate in triplicates and fluorescence reading was recorded using a microplate reader (Perkin Elmer, USA) (570 nm excitation and 600 nm emission wavelengths).