The membrane potential of C. sakazakii ATCC 29004 was determined according to the method described by Shi et al. [23 (link)]. Cells were diluted to 1 × 108 CFU/mL in PBS. Then, 125 μL cell suspensions were added into black 96-well microtiter plates (Nunc, Copenhagen, Denmark). Every cell suspension well was treated with the membrane-potential-sensitive fluorescent probe bis-(1,3-dibutyl barbituric acid) trimethine oxonol (DiBAC4(3); Molecular Probes, Eugene, OR, USA) for 30 min at 37 °C in the dark. The final concentration of DiBAC4(3) was 1 μmol/L. Then, 125 μL of LC-EO was added to wells to final concentrations of 0, 1, and 2MIC. With reference to the parameter setting of Shi et al. [23 (link)], the fluorescence of C. sakazakii was recorded by a fluorescence microplate reader (excitation 492 nm, emission 515 nm; SpectraMax M2, Molecular Devices, San Jose, CA, USA).
Black 96 well microtiter plates
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Black 96-well microtiter plates are a type of laboratory equipment used in various experimental procedures. They provide a standardized platform with 96 individual wells, each capable of holding a small volume of liquid sample. The black color of the plates helps to minimize well-to-well optical interference and improve the sensitivity of certain assays.
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2 protocols using black 96 well microtiter plates
1
Membrane Potential of C. sakazakii
2
Histochemical and Fluorometric GUS Assay
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A fragment of 2014 bp, upstream from the start codon in the gene At1G20080, was amplified using the primers listed in Supplemental Table 1 and cloned into the expression vector pCAMBIA1305.1, and then transformed into Arabidopsis by Agrobacterium tumefaciens. Homozygous lines were obtained and the histochemical GUS assays were performed as described (Jefferson et al., 1987) . Protein was extracted with 50 mM phosphate buffer (pH 7.3) supplemented with 10 mM EDTA, 0.1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 10 mM dithiothreitol. The fluorometric analysis reaction was performed in black 96-well microtiter plates (NUNC) in darkness at 37 C. The 50 ml of extraction buffer contained 1 mM 4-methylumbelliferyl-b-D-glucuronide as substrate and 1 mg of total protein. After 1 h, the reaction was stopped by addition of 150 ml of carbonate buffer (200 mM) and fluorescence intensity was measured using a multidetection Tecan Infinite M20 microplate reader (Tecan Trading AG) with 362 (±9) nm excitation and 450 (±20) nm emission. Protein concentration was measured using the Lowry-based Bio-Rad DC protein assay (Bio-Rad).
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