Elisa microplate reader

A previous study revealed that ATRA could facilitate the proliferation of H9c2 cells in normal condition [16 (link)]. In the present study, the effect of ATRA on H9c2 cell viability after H/R injury was evaluated using a WST-1 assay. Briefly, 1 × 10⁴ cells were seeded, per well, in 96-well culture plates (passage 6~10, ~3 × 10⁴ cells per cm²) and incubated for 24 h. ATRA, in 10-fold serial dilutions from 100 μM to 1 nM in DMSO, was added to the culture medium and allowed to incubate for 24 h. DMSO equivalent to ATRA at the highest concentration served as an H/R group. Control refers to cells with neither H/R injury nor ATRA treatment. After H/R treatment, 10 μM of WST-1 cell proliferation assay reagent was added to each well and allowed to incubate for 3.5 h (Roche Applied Science, Mannheim, Germany). Absorbance was read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (BioTek, VT, U.S.).

BV2 microglia cells were treated with lipopolysaccharide (LPS; 1 µg/mL) for 4 h, after which they were incubated with or without MCC950 for 2 h before stimulation with ATP (1 mM) for 24 h. Levels of IL-1β, IL-18, and TNF in the culture medium were measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol. Briefly, the 96-well microplates were coated overnight with anti-IL-1β, anti-IL-18, or anti-TNF antibody then blocked with 1% bovine serum albumin (BSA). Standards or culture medium (100 µL) were added to the microplates and incubated at room temperature for 2 h; this was followed first by incubation for 2 h with the biotin-conjugated detection antibody then by incubation for 30 min with 100 µL of streptavidin–horseradish peroxidase plus substrate for signal development. Finally, 100 µL of stop solution was added to each well and the OD₄₅₀ was measured using an ELISA microplate reader (Bio-Tek Instruments, Winooski, VT, USA) [30 ].

Embryos were removed from a pregnant ICR mouse at 14 days of gestation as described previously [13 (link)]. For neuronal viability assay, the cells were seeded in a 96-well plate at a density of 20,000 cells per well for 3 d. Then, the neurons were treated with 10 μM Aβ25-35 (Sigma-Aldrich, A4559) or cotreated with platycodigenin (Chengdu Desite Biotech Co. Ltd, Chengdu, China) for 3 days. The Aβ25-35 was previously incubated at 37 °C for 4 days for aggregation. Neurons were then incubated with 10 μL of CCK-8 solution (MCE, HY-K0301) for 3 h. The fluorescent absorbances were measured at 450 nm using an ELISA microplate reader (Biotek, Winooski, VT, USA). For normal neurite density measurement, the cells were seeded in an 8-well plate at a density of 10,000 cells per well for 2 d. Then the neurons were treated with PLA (0.01–10 μM) for 4 d. For Aβ treated neurite density measurement, the neurons were treated with 10 μM Aβ25-35 for 3 days before being treated with platycodigenin for 4 days. The cells were then prepared for immunocytochemistry.

The antibody titer of serums was identified by ELISA with RBMFP being coated as an antigen and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG MAb being used as a secondary antibody. First, purified RBMFP protein in carbonate coating buffer at a final concentration of 10 μg/mL was coated at 100 μL/well on the 96-well ELISA plates at 4°C overnight, and the unbound molecules were then washed 3 times by PBST. After a blocking step with 5% defatted milk at 37°C for 2 h and 3 PBST rinses were performed, serums from each week that were diluted in PBS with a 10 times gradient ratio (10² ~10⁷) were added at 100 μL/well and incubated at 37°C for 2 h. Then the plates were washed 3 times with PBST. After the wash step, the HRP-conjugated goat anti-mouse IgG MAb (1:5,000; MultiScience, Hangzhou, China) was added at 100 μL/well, reacted at 37°C for 2 h, and then rinsed with PBST. Then, the chromogenic tetramethylbenzidine (TMB; Innoreagents, Huzhou, China) solution was added at 100 μL/well and incubated at 37°C for 10 min. Finally, 50 μL/well of the ELISA stop solution (Beyotime, Beijing, China) was added, and the absorbance (optical density [OD]) at 450 nm was measured in a Bio-Tek ELISA microplate reader. The antibody titer of the serums was evaluated and statistically analyzed by GraphPad Prism 8.0 software.

Exudates from the animals’ air pouch were used to measure cytokines levels (IL-1β and TNF-α), using commercial Enzyme-Linked Immunosorbent Assay kits ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocol. Results were obtained using an ELISA microplate reader (Epoch, BioTek^®, Winooski, VT, USA) at 450 nm and expressed in ng/mL.

HUVECs were seeded at a density of 5 × 10⁴ cells/well in 24-well plate and incubated with various concentrations of glucose (5.5, 15, 30 and 60 mM) in the presence or absence of ADC or RES for 24-72 h. After treatment, the amount of LDH in the HUVEC culture media was determined according to the manufacturer’s instruction. The LDH activity was measured at 440 nm using an ELISA microplate reader (Bio-Tek Instruments).