Bio tek synergy h4 hybrid microplate reader

In total, 100 µL supernatant of sperm in Krebs solution was assessed by the determination thiobarbituric acid-reactive species (TBARS) assay; the method followed by Laoung-on et al. [5 (link)]. After the experimental procedure, the supernatant was investigated by a microplate reader (Bio Tek Synergy H4 Hybrid Microplate Reader, BioTek Instruments, Winooski, VT, USA) at 532 nm.

The thiobarbituric acid-reactive species (TBARS) assay was used to assess 100 μL of supernatant of homogenized left cerebral hemisphere. This method followed Laoung-on et al. [11 (link)], and then, the mixture solution was measured by a microplate reader at 532 nm (Bio Tek Synergy H4 Hybrid Microplate Reader, BioTek Instruments, Winooski, VT, USA).

A fluorescence-based potassium ion channel assay utilizes the ability of thallium (Tl⁺) to permeate K⁺ channels [18 (link)]. Once the K⁺ channels are open, Tl⁺ in the extracellular solution flows down its concentration gradient into the cells via K⁺ channels. Inside the cells, Tl⁺ binds to and activates a fluorogenic indicator dye preloaded into the cells, resulting in a dramatic increase in the fluorescence signal. This technique allows the rapid determination of K⁺ channel activity in a high-throughput manner [19 (link)]. We used a commercial kit (FluxOR Potassium ion channel assay, cat#: F10016, Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the manufacturer’s instructions to study the fluorescence detection of the Orf3a/E channel activity.
Samples of 10 μL each were added to a 96-well plate. Each sample was duplicated in two separate wells. The first replicate contained only the assay solution to establish the baseline signal and the second contained both the assay solution and Orf3a/E channel blockers. The fluorescence of each sample was repeated 3–6 times using a BioTek Synergy H4 Hybrid Microplate Reader (BioTek Instruments Inc. Winooski, VT, USA).