Glomax navigator microplate luminometer

SH-SY5Y cells were seeded with 4 × 10⁵ cells per well in six-well plates. The next day, cells were transfected with hsa-miR-34a-5p miScript miRNA Mimic or AllStars Negative Control (ANC) using HiPerFect Transfection Reagent (Qiagen, Hilden, Germany). At 24 h after transfection, cells were re-plated with 1.8–2.5 × 10⁴ cells per well in opaque-walled 96-well plates. After an additional 24 h, cells were treated with 1 µM tunicamycin or DMSO as control for 8 h, 24 h and 48 h. Luciferase-based CellTiterGlo 2.0, CytotoxGlo and Caspase3/7 Glo assays (Promega, Mannheim, Germany) were applied according to the manufacturer’s instructions to determine cell viability, cytotoxicity and caspase activity. Luciferase activity was measured with a GloMax navigator microplate luminometer (Promega, Mannheim, Germany). The experiments were done in triplicate in two independent experiments. As control, ANC and miR-34a-5p-transfected cells were treated with DMSO. Cell viability and caspase activity were calculated by normalizing the received luminescence values to the control transfected cells treated with DMSO for each time point. The relative number of dead cells was calculated by normalizing the luminescence derived from the dead cell protease activity to the protease activity of total cells after cell lysis. Statistical significance was calculated with a two-tailed paired t-test.

SARS-CoV-2 pseudotyped particles were produced by cotransfection of pSARS-CoV-2 S_trunc and pNL4-3ΔEnv-nanoluc in 293T cells (11 (link), 34 (link)). Fourfold serially diluted purified plasma IgG/IgA from COVID-19 convalescent individuals and healthy donors or monoclonal antibodies were incubated with the SARS-CoV-2 pseudotyped virus for 1 hour at 37°C. Subsequently, the mixture was incubated with Ace2-expressing cells for 48 hours. HT1080_Ace2 cl. 14 cells (34 (link)) were used for plasma-derived IgG or IgA assays, and 293T_Ace2 cells (11 (link)) were used for monoclonal antibody assays. After incubation, cells were washed twice with PBS and lysed with Luciferase Cell Culture Lysis 5× Reagent (E1531; Promega). NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (N1150; Promega) with the GloMax Navigator Microplate Luminometer (Promega). Relative luminescence units obtained were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of plasma-derived or monoclonal antibodies. The half-maximal and 90% inhibitory concentrations for purified plasma IgG or IgA or monoclonal antibodies (IC₅₀ and IC₉₀) were determined using four-parameter nonlinear regression (GraphPad Prism).

Firefly luciferase (fLuc)-expressing MLV pseudovirus carrying the S protein of SARS-CoV-2 (D614G variant of an early 2020 isolate with NCBI accession number YP_009724390.1) was produced as reported^{13 (link)}. Briefly, HEK293T cells in 6-well plates were transfected with three plasmids encoding MLV gag-pol and fLuc reporter (kind gift from S. Pöhlmann, Germany) plus the S protein. After three days incubation at 33 °C, the pseudovirus-containing supernatants were harvested and frozen. For the entry inhibition assay, Vero cells or A549-AT cells (i.e. A549 cells expressing ACE2 and TMPRSS2; code a549d-cov2r from Invivogen) were seeded at 7500 cells per well in white half-area 96-well plates. One day later, the cells were pre-incubated with serial compound dilutions (in serum-free medium) for 20 min at 37 °C, after which the pseudovirus was added and spinoculated by centrifugating the plates at 453 × g for 45 min at 37 °C. After placing the plates for two more hours in the incubator, the supernatants were removed and replaced by medium with 10% FCS. After 3 days incubation at 33 °C, fLuc activity was monitored using a luciferase assay system kit and GloMax® Navigator Microplate Luminometer (both from Promega). The EC₅₀ values of the compounds were calculated with Graphpad software, as explained in Section “Anti-coronavirus activity and cytotoxicity in CPE reduction assays”.

The cells were incubated with CellTiter-Glo Reagent (Promega) with the same volume as the cell culture media, then mixed for 2 min on a shaker. The cells were then placed for 10 min at room temperature. Next, the mixture was moved to opaque-walled multi-well plates, then the signal was measured using GloMax Navigator Microplate Luminometer (Promega).

Primary cultures of Bdnf-Luc mouse cortical cells at 13 DIV were treated with 25 mM KCl (for 6 h), a series of compounds (for 6 h), or herbal extracts (6, 24, or 48 h). Primary cultures of Bdnf-Luc mouse cerebellar granule cells at 7 DIV were treated with 25 mM KCl (for 6 h). Luciferase activity of each well was measured using the Steady-Glo Luciferase Assay System (Promega) with a Glo-Max Navigator microplate Luminometer (Promega), according to the manufacturer’s instruction. White adhesive seal was added to the bottom of the microplate (Perkin Elmer, Waltham, MA, USA) to make it opaque before the measurement of luciferase activity.

To study the effect of yqiC on ATP production, an ATP assay of the overnight cultures of the S. Typhimurium SL1344, ΔyqiC, and ΔyqiC' strains was performed using a BacTiter-Glo Microbial Cell Viability Assay (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. Finally, a GloMax Navigator Microplate Luminometer (Promega) was used to read the 96-well plates containing the samples, and their luminescence was recorded. The experiments were performed independently in triplicate. The generated ATP concentrations of ΔyqiC were statistically compared with those of SL1344 and ΔyqiC' by using Student’s t test. A p value of < 0.05 was regarded as statistically significant.