The fluorescence nuclease activity assays were performed in reaction volumes of 10 μL, by mixing 8 μL of sample (24 h culture supernatants or control medium) with 1 μL of CaCl2 100 mM and 1 μL of F-TTprobe (50 pmol·μL−1). The reactions were incubated at 37 °C, for 30 min. Then, the reactions were stopped by adding 295 mL of PBS-/- supplemented with 10 mM EDTA. Finally, 95 mL of each sample was loaded in triplicate into 96-well black plates (96F non-treated black microwell plate, Thermo Scientific). Fluorescence intensity was measured with a fluorescence microplate reader, Synergy Neo2 Hybrid Multi-Mode Reader from Agilent BioTek (Winooski, VT, USA) using the filter settings for FAM (excitation/emission 485/528 nm). Three independent experiments were performed with each sample. The results are expressed as the mean ± SD (n = 3) of fluorescence intensity in arbitrary units (a.u.).
96f non treated black microwell plate
Manufactured by Thermo Fisher Scientific
The 96F non-treated black microwell plate is a laboratory consumable item designed for various scientific experiments and assays. It features a 96-well format with a flat bottom and a black color. The plate is non-treated, meaning it does not have any special coatings or surface modifications.
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3 protocols using 96f non treated black microwell plate
1
Nuclease Activity Assay Protocol
2
Nuclease Activity Assay Protocol
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Nuclease activity assay was performed as previously reported [49 (link)], with small modifications. Specifically, for each reaction, 5 μL of sample (HB or samples homogenates) was combined with 4 µL PBS (+/+) and 1 μL (50 pmoles) of oligonucleotide probe (nuclease substrate) and incubated at 37 °C, for 1 hour. After the incubation period, the reaction was stopped by adding 295 μL of PBS (-/-) supplemented with 10 mM EDTA. Next, 95 μL of each sample was loaded in triplicates into 96-well plates (96F non-treated black microwell plate, Thermo Fisher Scientific). Fluorescence intensity was measured with a fluorescence microplate reader (Synergy HT, BioTek, Winooski, VT, USA), using the filter settings for FAM (excitation/emission (ex/em), 494/521 nm)).
3
Nuclease Activity Assay Protocol
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The nuclease activity assays were performed using the standard conditions previously reported [15] . Specifically, for each reaction, 9 μL of sample (i.e. culture supernatants of Salmonella, other bacteria or mesenteric lymph nodes, control medium or buffer) were mixed with 1 μL (50 pmol, nuclease substrate) of the oligonucleotide probe to be tested and incubated at 37°C, for 1 h. Thereafter, the reaction was stopped by adding 295 μL of PBS -/supplemented with 10 mM EDTA. Next, 95 μL of each sample was loaded in triplicates into 96-well black plates (96F non-treated black microwell plate, Thermo Scientific). Fluorescence intensity was measured with a fluorescence microplate reader (Synergy HT, BioTek) using the filter settings for FAM (excitation/emission 494/521 nm). Three independent experiments were performed with each sample. The results were expressed as the mean ± SD (n=3) of fluorescence intensity in arbitrary units (a.u.).
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