Soluble protein concentrations of coral tissue samples were quantified using a colorimetric protein assay kit (Pierce BCA Protein Assay Kit; Glasl et al., 2019b (link)). Tissue pellets were thawed on ice and resuspended in 1 ml PBS. The resuspension (25 µl) was added to 200 µl of working reagent from the kit in a 96-well plate. The plate was mixed thoroughly on a plate shaker for 30 s and then incubated at 37 °C for 30 min. The plate was cooled down at room temperature. The absorbance was measured at 563 nm in a Cytation 3 multi-mode microplate reader (BioTek, Winooski, USA) and analyzed using the software Gen5 (BioTek, Winooski, USA). Measurements of the standards and samples were blank corrected to remove background absorbance. For each plate, a protein standard curve was obtained using bovine serum albumin (BSA) solution at concentrations between 25 and 2,000 µg ml−1.
Cytation 3 multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Cytation 3 is a multi-mode microplate reader from Agilent Technologies. It is designed to perform absorbance, fluorescence, and luminescence measurements on microplates.
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Lab products found in correlation
Lysis buffer, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Fluorometric assay kit, by Abcam (1 mentions)
Prism 7, by GraphPad (1 mentions)
Nanodrop nd 100 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cignal antioxidant response reporter luc kit, by Qiagen (1 mentions)
Cellevent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
10 protocols using cytation 3 multi mode microplate reader
1
Quantification of Coral Tissue Protein
2
Wnt/β-catenin Signaling Pathway Assay
3
Quantitative Screening of Quorum Sensing Inhibitors
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A modified method of Bjarnsholt et al. (2005) (link) was performed for the determination of QSI potentials of ethyl acetate CE and CFSE from extremophilic N. versiforme. QSI screenings were performed in 96-well black microplates (Nunc, Thermo Scientific). 100 μl of M9 medium was added to each well and two-fold serial dilutions of tested extracts were made by adding 100 μl to the first well and after mixing, transferring 100 μl from first to the next well and so on. Overnight cultures of the lasB-gfp, rhlA-gfp, and pqsA-gfp monitor strains were added to obtain a total volume of 200 μl with an OD 450 nm of 0.1. Final concentrations of tested extracts were 240 μg/ml, 120 μg/ml, and 60 μg/ml. The tests were performed in three replicates. Bacterial growth and GFP expressions were monitored using Cytation 3 multimode microplate reader (BioTek, United States) for 14–16 h, measuring absorbance and fluorescence every 15 min. Fluorescence of GFP expression was measured at 485 nm excitation and 535 nm emission wavelengths.
4
Quantification of Coral Chlorophyll a
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Photopigment (chlorophyll a) concentrations in the tissue of corals were quantified using a spectrophotometric approach (Glasl et al., 2019b (link)). Tissue pellets were thawed on ice to avoid sample degradation and resuspended in 1 ml of 90% ethanol. Samples were sonicated for 1 min and centrifuged for 5 min at 10,000 rcf. Subsequently, 700 µl of the supernatant was removed and transferred to a new tube. The resuspension, sonication and centrifugation were repeated on the remainder of the pellet. The supernatant was recovered again, combined with the previous extraction and mixed by inversion. Sample extract and 90% ethanol (blank read) were loaded in triplicate (200 µl each) to a 96-well plate and the absorbance was recorded at 470, 632, 649, 665, 696 and 750 nm in a Cytation 3 multi-mode microplate reader (BioTek, Winooski, USA) and analyzed using the software Gen5 (BioTek, Winooski, USA). Blank corrected absorbance measures were used to calculate chlorophyll a concentrations (Equation S1).
5
Enzymatic Activity Assay of Purified PvLDH
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Enzymatic activity of the purified PvLDH was determined with a fluorometric assay kit (Abcam, Cambridge, UK) following the manufacturer’s protocol. Briefly, 100 µL of assay mixture per sample was added to each well of black microplate and the reaction was performed at 37 °C for 30 min. The assay mixture consisted of 45.5 µL of LDH assay buffer, 2.5 µL of the PicoProbeTM, 2 µL of substrate, and 50 µL of PvLDH at various concentrations. At this time, PvLDH solutions were prepared at various concentrations (0, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.50, 5.00, 10.00 nM) using two-fold serial dilution. The reaction was performed in triplicate at these 11 different concentrations. BSA was used as a negative control and reacted under the same conditions. Finally, fluorescence intensity was measured using a Cytation3 multimode microplate reader (BioTek, Winooski, VT, USA). The excitation and emission wavelengths were 535 and 587 nm, respectively. Based on the measured fluorescence intensity values, a dose–response curve was prepared using GraphPad Prism 7.0.
6
Bacterial Reporter Assays with C4-HSL
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Strains containing the reporter fusions were grown overnight in TSB with appropriate antibiotics and diluted at an OD600 of 0.05 in TSB. For lacZ reporter assays, culture samples were regularly taken for determination of growth (OD600) and β-galactosidase activity (48 ). For lux reporter assays, luminescence was measured using a Cytation 3 multimode microplate reader (BioTek Instruments, USA). When mentioned, C4-HSL was added at a final concentration of 20 μM from a stock solution prepared in high-performance liquid chromatography (HPLC)-grade acetonitrile. Acetonitrile only was added in controls. All OD600 measurements were performed with a NanoDrop ND100 spectrophotometer (Thermo Fisher Scientific, Canada).
7
Catalase Activity Assay using H2O2
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The assay consists in the reaction of 20 μL of sample with 100 μL of H 2 O 2 solution in a 300 μL final reaction volume. Each well was prepared according to Table 2 and Fig. S1. Catalase activity was measured by adding H 2 O 2 to the samples and following its decomposition over time by 240 nm absorbance (Aebi, 1984) . To ensure that the method is working properly and is specific for catalase dependent H 2 O 2 degradation, several controls were performed (Table 2).
A 240 nm initial basal absorbance was determined for background correction and protein loading assessment. Subsequently, 100 μL of phosphate buffer 50 mM, pH = 7.8 (PB) was added to columns 1 and 12 (Table 2 last line). Next, using a multichannel, 100 μL of H 2 O 2 solution 90 mM was added simultaneously to columns 2-11 immediately before starting the kinetic reading. Catalase activity was followed by the variation of H 2 O 2 absorbance at 240 nm during 2.5 min, with a readout each 15 s using a Cytation3 multi-mode microplate reader (BioTek Instruments, Inc.,Winooski,VT,USA). A typical catalase reaction/H 2 O 2 degradation is shown in Fig. 1A.
A 240 nm initial basal absorbance was determined for background correction and protein loading assessment. Subsequently, 100 μL of phosphate buffer 50 mM, pH = 7.8 (PB) was added to columns 1 and 12 (Table 2 last line). Next, using a multichannel, 100 μL of H 2 O 2 solution 90 mM was added simultaneously to columns 2-11 immediately before starting the kinetic reading. Catalase activity was followed by the variation of H 2 O 2 absorbance at 240 nm during 2.5 min, with a readout each 15 s using a Cytation3 multi-mode microplate reader (BioTek Instruments, Inc.,Winooski,VT,USA). A typical catalase reaction/H 2 O 2 degradation is shown in Fig. 1A.
8
Dual-Luciferase Reporter Assay
9
Evaluating Apoptosis via Caspase 3/7
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The activation of caspase 3/7 was assessed to evaluate if the cytotoxic core of MCTs carry features of apoptosis. The CellEvent™ caspase-3/7 green reagent is intrinsically non-fluorescent, as the DEVD peptide inhibits binding of the dye to DNA. However, upon activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, and the free dye can bind the DNA, generating a bright green fluorescence. The CellEvent™ (Thermo Fisher Scientific R37111) ready to use reagent (25 µl) was added to each well containing spheres on day 4. After 30 min of incubation at 37 °C, images were taken using the Bio-Tek Cytation3 Multi-mode microplate reader (Agilent).
10
Fluorescent Labeling of Cytoskeletal Actin
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A stock solution of Alexa Fluor® 594 Phalloidin (Life Technologies, Carlsbad, CA, USA) was diluted from its 6.6 μM at a 1:40 ratio in PBS containing 1% BSA. The cells were incubated for 20 min at RT with a solution containing 5 µL of stock Phalloidin in 200 µl of PBS. At the end of the incubation the staining was removed, and the cells were washed twice with PBS. Thereafter, 200 µL of 300 nM DAPI (Life Technologies) solution in PBS was added. Cells were incubated with DAPI staining for 10 min, washed twice with PBS, and kept at 4 °C in PBS until imaging. Images were taken using the Bio-Tek Cytation3 Multi-mode microplate reader (Agilent).
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