Elx808 microplate reader

The heterologous expression of A. baumannii genes was performed in S. Typhimurium to test EAL, NLA, and ACAT functions. NB was inoculated with a single colony, and the colony was grown overnight at 37°C. The following day, 1% (vol/vol) of overnight culture (~16 h old) was transferred into minimal medium in a 96-well microtiter plate. S. Typhimurium growth experiments were performed in NCE minimal medium (41 (link)) containing Wolfe’s trace minerals (46 (link)), MgSO₄ (1 mM), glycerol (22 mM), and ampicillin (100 μg/mL). Cobamide precursor salvaging was performed by assessing growth with the addition of dicyanocobinamide [(CN)₂Cbi] and 5,6-dimethylbenzimidazole (DMB) (150 μM). Gene expression was induced with l-(+)-arabinose (0.5 μM). The growth of cultures in microtiter plates was monitored every hour at 630 nm during incubation with shaking at 37°C in a BioTek Elx808 microplate reader. To test the functionality of the A. baumanniieutBC gene products, minimal medium containing no-carbon nonessential NCE medium (47 (link)) with ethanolamine (30 mM) provided as the sole source of nitrogen was used. In each case, growth analysis was performed in a BioTek Elx808 microplate reader with three biological replicates in technical triplicate.

To determine the seroprevalence, we utilized an in-house ELISA test that we recently developed and validated [9 (link)] to measure SARS-CoV-2 IgM and IgG antibodies against the most immunogenic SARS-CoV-2 antigens, nucleocapsid protein (N) and spike glycoprotein (S). Briefly, commercial recombinant SARS-CoV-2 S1 subunit (amino acids 1–685) (Sino Biological, Beijing, China), and an in-house produced recombinant SARS-CoV-2 N protein were used to coat 96-well ELISA plates at 1 μg/mL and 4 μg/mL in phosphate-buffered saline (PBS), respectively, for overnight at 4 °C. Plates were then washed with PBS with 0.05% tween-20 (PBS-T). After blocking the plates with 5% skim milk in PBS-T buffer at 37 °C for 1 h, 1:100 diluted serum samples were incubated for 1 h at 37 °C and washed. Anti-IgG and anti-IgM HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were then added for 1 h at 37 °C. Following incubation, plates were washed and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD, USA) was added for 30 min in the dark at room temperature, and the reaction was terminated by 0.16 M sulfuric acid. Absorbance was measured at 450 nm using the ELx808 microplate reader (BioTek, Winooski, VT, USA). Cutoff values for the ELISA were 0.4 for IgG N-ELISA, 0.55 for IgM N-ELISA, 0.17 for IgG S1-ELISA, and 0.3 for IgM S1-ELISA as previously determined [9 (link)].

WST-1 is a water-soluble tetrazolium salt that is cleaved to a formazan dye in a mechanism mainly dependent on NAD(P)H production by metabolically active cells. 2 days after transfection, 1 X 10⁴ cells (2 X 10⁴ for H522) were seeded in triplicate for each sample in clear flat bottom 96 well plates, and left for 3 days before performing the assay according to manufacturer’s instructions (Sigma-Aldrich). Briefly, 10 μl Cell Proliferation Reagent WST-1 was added to each well containing 100 μl media and incubated for 30 min to 4 h. Absorbance values (that ranged between 0.5–2) were determined on an ELx808 microplate reader (BIO-TEK Instruments) at 450 nm against a blank control background. Cell proliferation (%) was determined by calculating (mean absorbance of sample / mean absorbance of control) X 100. 2-day VP-16 (Sigma-Aldrich) treatments were used as positive controls.