96 well black microplate

The Pla activity was evaluated using a fluorometric assay as previously described [46 (link), 47 (link)]. Y. pestis strains were grown on HIB agar at 26°C for 36 h, replated to fresh HIB, and incubated at either 26°C or 37°C for 24 h. Bacteria were harvested from the plates, suspended in PBS and adjusted to optical densities (OD₆₀₀) of 0.1 (5 × 10⁷ cfu/ml) and 0.05 (2.5 × 10⁷ cfu/ml) for the cultures grown at 26°C and 37°C, respectively. The bacterial titers were verified by plating the suspensions on HIB agar in 10-fold dilutions. The assay was conducted with 50 μl of bacterial suspension containing 2.5 μg of the fluorescently labeled hexapeptide substrate DABCYL-Arg-Arg-Ile-Asn-Arg-Glu (EDANS)-NH₂ [48 (link)] in the black 96-well microplate (Costar Corning Inc., Corning, NY) in quadruplicates. The kinetics of substrate cleavage by the Pla expressed on the surface of Y. pestis cells was measured every 10 min for 6 h at 37°C by using a BioTek Synergy HT reader (BioTek Instruments Inc., Winooski, VT) at excitation/emission wavelength of 360 nm/460 nm.

The oxygen radical absorbance capacity (ORAC assay) was analyzed according to Huang, Ou, M., Flanagan and Prior [64 (link)] and Ganske [65 ] with slight modifications. Approximately 60 µL of 10 mM fluorescein was added into a black 96-well microplate (Costar, cat no. 3694) along with 10 µL of sample (seaweed extract), standard (Trolox) or water (blank). Approximately 40 µL of phosphate buffer solution (pH 7.4) was added in one well for gain adjustment. The mixture was incubated at 37 °C for 10 min. After incubation, 30 µL of 120 mM 2,2 azobis (2-methylpropionamidine) dihydrochloride solution (AAPH solution) was added and the fluorescence emission (excitation at 485 nm, emission at 520 nm) was read every minute for 100 min in a POLARstar optima fluorescence analyzer (BMG Labtech, Ortenberg, Germany). The area under the fluorescence curve (AUC) was calculated by the normalized curves. Each sample was measured in triplicate.

A detergent screen was performed for the hTRPM4-eGFP fusion protein, where 5 mL pellets of frozen baculovirus infected cells were solubilised in several different detergents including 1% Fos-choline-14, 2% DDM, 1% DM, 1% Lauryl Maltose Neopentyl Glycol (LMNG), 1% digitonin and a mixture of 0.5% DDM plus 0.5% LMNG (wt/vol) dissolved in a base buffer containing (40 mM Tris pH 7.5, 150 mM NaCl, 150 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 6 mM β-mercaptoethanol, 5 mM L-Arginine and 10% glycerol). All cell pellets were solubilised for 2 h at 4 °C with rotation before being subjected to fluorescence size-exclusion chromatography (FSEC) to determine the size homogeneity of the proteins after solubilisation (whether they be aggregated, oligomeric or degraded). FSEC was performed on an AKTA purifier system (Amersham Biosciences) using a Superose 6 10/300 GL column (GE Healthcare) pre-equilibrated with the base buffer and 0.01% (wt/vol) of the detergent the cell pellet was solubilised in. 1 mL of the solubilised material was injected per run onto the Superose 6 column and fractions were collected. Samples from individual fractions (80 μL each) were aliquoted into in a black 96 well microplate (Corning) and scanned for fluorescence using a BMG PHERAstar FS multimode plate reader (BMG Labtechnologies, Durham, NC).

The cellular reactive oxygen species level in L. minor was quantified using the 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) assay (Molecular Probes Inc., Eugene, OR, USA), as originally developed by Razinger et al. (2010 (link)) and further modified for L. minor by Xie et al. (2018 (link)). When binding to ROS (predominantly hydrogen peroxide), non-fluorescent H₂DCFDA will be oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF) with excitation around 488 nm (Kaur et al. 2016 (link)). In the present study, a 50 mM H₂DCFDA stock solution was prepared in dimethyl sulfoxide (DMSO, purity 99.7%; Sigma-Aldrich, St. Louis, MO, USA) and stored at − 20 °C until use. After the exposure, fronds were immersed in 200 µL working solution of H₂DCFDA (50 µM) prepared in the culture medium. After 1 h of probe loading, the fronds were rinsed with clean medium and transferred to a black 96-well microplate (Corning Incorporated, Costar^®, NY, USA). The fluorescence of the fronds was immediately measured using a VICTOR³ fluorescent plate reader, 1400 Multilabel Counter (Perkin Elmer, Boston, MA, USA) with excitation/emission wavelength of 485/538 nm. The background fluorescence of the medium-probe mix (without the presence of fronds) was also measured and subtracted from the total fluorescent counts of the samples.

The potassium-sensitive benzofuran isophthalate probe (PBFI; Invitrogen, Carlsbad, CA, USA) was used to determine the release of potassium ions from desiccation-adapted Salmonella Tennessee as described previously [9 (link),11 (link)]. Briefly, aliquots (1 mL, each) of desiccation-adapted cells in saline (Figure 1) were harvested by centrifugation at 8000× g for 5 min, and cell pellets were resuspended in 5 mM HEPES buffer (Sigma Aldrich) containing 5 mM glucose (Fisher Scientific, Fair Lawn, NJ, USA). Portions of these cell suspensions (90 µL) were added to wells of a black 96-well microplate (Corning) followed by the addition of the PBFI probe at a final concentration of 2 µM. Then, 10 µL of carvacrol or thymol was added to each well to achieve final concentrations of 80 to 800 µg/mL of each compound, and polymyxin (10 µg/mL) served as a positive control. Concentrations of released potassium ions were measured as changes in fluorescence using a microplate reader (PerkinElmer, Wellesley, MA, USA) at excitation and emission wavelengths of 346 and 505 nm, respectively. Fluorescence measurements were captured every 100 s and normalized by subtracting the background noise.