96 well black plate

To conduct kinetics assay in separated incubation, prepared 44.3 mL of Aβ_1–42 dimethyl sulfoxide (DMSO) stock solution (5 mM) was diluted with 4.4 mL PBS (20 mM, pH = 7.4), (working concentration: 50 μM of Aβ_1–42 containing 1% DMSO). Five sets of diluted solutions were incubated in an orbital shaker (37 °C, 300 rpm); 2970 μL of the incubated solution was collected every 30 min for 6 h and then at 12, 24, 36, 48, 60, and 72 h. Then, 30 μL of Q-OB DMSO solution (100 μM) (or 1 mM of ThT DMSO solution) was added to the collected solution, and the fluorescence spectra were recorded (λ_ex = 460 nm for Q-OB and λ_ex = 440 nm for ThT) (slit 2.5/2.5, n = 3 independent experiments).
To conduct kinetics assay in coincubation, Aβ_1–42 and probe (Q-OB or ThT) stock solutions were diluted with PBS (20 mM, pH = 7.4), aCSF (Tocris Bioscience), or DW [working concentration: 10 μM of Aβ_1–42 and 1 μM of Q-OB (or 10 μM of ThT), respectively, containing 1% DMSO]. The mixture solution (100 μL) was transferred to a 96-well black plate (SPL, Korea) and covered with adhesive optical sealing film (Bioneer Inc., Korea). Fluorescence intensity was measured using a Hidex Sense Microplate Reader every 10 min under orbital-shaking incubation for 12 h (37 °C, 200 rpm) (λ_ex = 460 nm, λ_em = 580 nm for Q-OB, and λ_ex = 440 nm, λ_em = 490 nm for ThT) (n = 3 independent experiments).

Freeze CSF aliquots were thawed at 0 °C for 30 min, followed at 25 °C for 10 min, and used without a pre-treatment procedure. Prepared Aβ_1–42 and Q-OB stock solution in DMSO were diluted with CSF (working concentrations: 10 μM Aβ_1–42 and 1 μM Q-OB in CSF containing 1% DMSO) and treated with a vortex to be evenly dissolved. Then, the Q-OB-treated CSF was transferred to a 96-well black plate (SPL, Korea) (100 μL per well) and covered with an adhesive optical sealing film (Bioneer Inc., Korea). Fluorescence intensity was measured using a microplate reader every 10 min under orbital shaking incubation for 12 h (37 °C, 200 rpm) (λ_ex/λ_em = 460/580 nm) (n = 3 independent experiments).

For the analysis of phagocytosis, BMDMs (1 × 10⁴ cells per well) plated in a 96-well black plate (SPL Life Science Co.) were incubated with 0.005% fluorescent carboxyl-modified polystyrene latex beads (30 nm [L5155] or 1 μm [L4655], Sigma-Aldrich Co.) in the presence of MB (0, 20, 100, or 200 μM) for 6 h. After washing three times with PBS, fluorescence intensity was measured using a plate reader (470/505 nm, Synergy™ H1 Hybrid Multi-Mode Reader, BioTek).

Arabidopsis leaf discs (4 mm diameter) were transferred to water in a 96‐well black plate (SPL Life Sciences Co. Pocheon‐si, South Korea) and incubated at room temperature overnight. Before the induction of ROS, the normalized leaf discs were floated on C. fusca culture, C. fusca supernatant, 10 nm lactic acid, and BG11 broth in a 96‐well plate for 1 h and then washed twice with sterile distilled water. To trigger ROS production, leaf discs were placed in 100 μl of assay solution containing 10 ng ml⁻¹ peroxidase, 20 μm luminol, and 100 nm flg22 peptide. Light emission was measured as RLU using a Mithras Tristar2 LB 940 (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany), which is a 96‐well luminometer, for 1 h.

To examine the protein interactions of SRD3c with HDAC4c or HDAC5c mutants in living cells, bimolecular fluorescence complementation (BiFC) assays were carried out using a Fluo-Chase kit (Amalgaam), according to the manufacturer’s manual. Briefly, SRD3c and HDAC proteins were fused to the N- or C-terminal portions of Kusabira Green protein, resulting in KGN-SRD3c and KGC-HDAC4c or -5c constructs, respectively. The KGN-SRD3c was coexpressed with KGC-HDAC4c or -5c in HEK293 cells using the SuperFect system in a 96-well black plate (SPL life science). After 48 h of transfection, the fluorescent signals (excitation wavelength: 494 nm, emission wavelength: 538 nm) from the cell lysates were measured using a fluorescence spectrophotometer (Molecular Devices, Spectra max GEMINIXPS). The quantitation experiments were repeated two times for the triplicated samples.

PDEδ was diluted to make a series of two-fold dilutions with a starting concentration of 32 µM. The diluted solution was mixed with PBS buffer [137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ (pH 7.2)] and then the mixture was loaded on a 96 well black plate (SPL life Science, Gyeonggi-do, Republic of Korea). Compounds were transferred to the wells of assay plate and the final concentration of compounds was fixed to 0.5 µM. After the addition of the compound, the assay plates were incubated for 2 h at 4 °C. FP values were detected by SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA) at the maximum absorption and emission wavelengths of PD compounds in assay buffer. K_d values were determined by previously reported method³⁷ (link).

HUVECs were seeded at a density of 2 × 104 cells per well in a 96-well black plate (SPL Life Sciences, Pochun, Gyeonggi-do, South Korea) and incubated in humidified 5% CO₂ incubator at 37˚C. The culture medium was changed to FBS-free culture medium after 24 h, and ginseng berry extract at various concentrations (20, 100, and 500 μg/ml) was added to each well. TNF-α (10 ng/ml) was added after 30 min. Detection of intracellular NO was performed according to the kit, OxiSelectTM Intracellular Nitric Oxide (NO) Assay Kit (Cell Biolabs, San Diego, CA, USA) manual and fluorescence was measured with a fluorometric plate reader at 480 nm/530 nm.