Enspire

Total cellular ATP content was measured using an ATPlite assay kit (PerkinElmer) according to manufacturer’s instructions. Briefly, 15 000 cells were counted and used to evaluate ATP content in iPSC, whereas 20 000 neuron progenitor cells were seeded in a 96-well plate and cultured as normally until day 30, when cells were lysed on the plate prior the assay. Six wells per cell line were used. Luminescence was monitored with an EnSpire (PerkinElmer) microplate reader with 0.1 s measurement time and normalized to the cell count (iPSC) or protein levels (MN). Intracellular and extracellular lactate levels were measured using Amplite™ Colorimetric L-Lactate Assay Kit (AAT Bioquest) according to the manufacturer’s protocol. Briefly, 200 000 neuron progenitors were seeded and cultured on 12-well plates and cultured as normally until day 30. Then, 200 μl of the media was used for the extracellular lactate measurement and cells were harvested and lysed for the intracellular lactate measurement. Three wells per cell line were used. Extracellular samples were diluted 1:10 in PBS and all samples were filtered through a 10 kDa MW spin filter (Millipore, Darmstadt, Germany). Fluorescence was recorded at Ex/Em = A540 nm/A590 nm using an EnSpire (PerkinElmer) microplate reader and normalized to protein levels.

Primary hippocampal culture was prepared [46 (link)] as previously described. Primary hippocampal neurons cultured in 6-well plates were underwent OGD or not (naïve group) after 5–6 days in culture [19 (link)]. To obtain the extract, the BYHW decoction was suspended in HBSS at a concentration of 35 mg/mL, mixed thoroughly with a vortex for 5 minutes, and centrifuged at 150 g for 30 minutes to collect the supernatant. Mitochondria damage, apoptosis, ROS, and ATP content were detected using MitoTracker kit, Annexin V-FITC/PI kit, MitoSOX red kit, and ATP content kit according to the instruction of the manufactures, respectively. Damage to the mitochondria and neuronal apoptosis was measured in a BD FACSAriaTM llu flow cytometer and analyzed using the DIVA software from BD Biosciences (San Jose, CA, USA). ROS in cells were evaluated by fluorescent microplate reader (PerkinElmer EnSpire™, USA). ATP content was measured (PerkinElmer EnSpire™, USA) at 340 nm immediately and after 3-minute incubation at 37°C. All experiments were performed at least three times.

The concentrations of 4-hydroxynonenal (4-HNE) were measured with a 4-HNE ELISA kit (MLBio, Inc., Shanghai, China) according to the protocol. Samples absorbance was measured at 450 nm with a microplate reader (Enspire; PerkinElmer) and calculated according to a standard curve. The GSH/GSSG ratio was determined by a commercial detection kit (Nanjing Jiancheng Bio-engineering, Inc., Nanjing, China) according to the manufacturer’s protocol. The optical density value of sample was determined at a wavelength of 405 nm with a microplate reader (Enspire; PerkinElmer).

With reference to the method described previously (Katharine and Watnick, 2003 (link)), wild strains HY9901 and HY9901 ΔtyeA (OD₆₀₀ = 0.5) were transferred to a 96-well plate. Each well was inoculated with 200 μL of bacterium with 6 replicates per sample, the negative control was an inoculum of TSB only, and the culture temperature was 28°C. Samples were taken from the 96 well plate at 12, 24, 48, and 72 h, methanol fixed for 20 min, stained with Crystal violet ammonium oxalate dye for 15 min, rinsed with water and dried. Finally, 95% alcohol was added and incubated at room temperature for 30 min. OD₅₇₀ was determined by Multimode Plate Reader(PerkinElmer EnSpire, EnSpire, Singapore) (OD₆₀₀ = 1, bacterial concentration = 1 × 10⁹ cfu/mL).

The amount of adsorbed proteins on NP surface was determined by BCA assay (Sigma Aldrich). Briefly, 5 × 10⁴ cells were seeded on a 35 mm cell culture dish and incubated for 24 h with NP suspensions to allow endocytosis. Conversely, for NP shooting, cells were shot with NPs by gene gun method. After 24 h, endo and shot cells were washed roughly with PBS, trypsinized, counted, and seeded at a density of 7 × 10⁴ cells/cm² on a Transwell filter with a pore size of 4 µm and incubated for 72 h at 37 °C. The Transwell filters were pre-treated with rat tail type I collagen for 1 h at 37 °C in a dry incubator in order to allow the cellular adhesion. Afterward, the basal medium was recovered, centrifuged, and the pellet was rinsed twice with PBS in order to remove cellular debris. Next, the pellet was suspended in 300 µL of sterilized water and was analyzed using a spectrofluorometer (EnSpire, Perkin-Elmer) in order to quantify the amount of recovered NPs. BCA assay was performed using the same number of NPs for endo and shot NPs according to the manufacturer’s procedure (Sigma Aldrich). Absorbance of BCA assay reagent solution was read at 560 nm by a plate reader spectrophotometer (EnSpire, Perkin-Elmer). Data were reported as µg/mL of protein normalized with non-conditioned NPs.

The cells were scraped down from the bottom of 24-well plates and collected by centrifuging at 2000 rpm for 5 min. The cell pellet was resuspended with 0.4 mL of 5% trichloroacetic acid (TCA) and ultrasonic decomposed in an ice-water bath for 1.0 min with 2 s on and 2 s off. The cell lysate was centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatant was used for GSH and GSSG assays. 3.6 mL phosphate-EDTA buffer (pH 8.0) and 200 μL OPA (1 mg/mL) was added into 200 μL of the supernatant and incubated for 40 min at room temperature. Fluorescent intensity was measured using a automatic microplate reader (PerkinElmer EnSpire) at an excitation wavelength of 350 nm and an emission wavelength of 425 nm. For the GSSG analysis, 40 μL NEM (0.04 mol/L) was added to another 100 μL of supernatant and incubated for 30 min at room temperature. 1.9 ml of NaOH (0.1 mol/L) and 100 μL of OPA (1 mg/mL) were added into the mixture and incubated for another 15 min at room temperature. Fluorescent intensity were measured with excitation wavelength of 337.8 nm and emission wavelength of 421.6 nm on an automatic microplate reader (PerkinElmer EnSpire). The concentrations of both GSH and GSSG were determined by standard curves of GSH and GSSG.