Spectramax i3x plate reader

Steady-state FP recordings were performed
using a SpectraMax i3x
plate reader (Molecular Devices).^{61 (link),76 (link)} All steady-state
FP measurements were conducted using a buffer that contained 20 mM
Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.005% Tween 20,
and 96-well black untreated polystyrene microplates (Corning Inc).
Other details of steady-state FP measurements were previously reported.^{44 (link)} 100 μL of each 20 nM labeled SET1_Win peptide was added to individual wells at a final concentration
of 10 nM. The steady-state FP anisotropy was measured on the plates
after a 1 h incubation at room temperature in the dark. WDR5-dependent
dose-response data were averaged and then fitted using a four-parameter
logistic function to acquire the binding affinity (K_D) for each interaction pairs.

Lyophilized pigment toxin or control extracts were dissolved in DTS to a final concentration of 200 μM and were incubated overnight at room temperature and protected from light before use. To perform the hemolytic titer, twofold serial dilutions of purified pigment in DTS from A909 WT or L. lactis pcylX-K were performed in PBS in a final volume of 100 μL. Twofold serial dilutions of control extracts (from GBSΔcylE or L. lactis pEmpty) in DTS were also performed in PBS as controls. The samples were incubated with 100 μL of EDTA-treated human red blood cells (0.5% in PBS) in 96-well plates for 1 h at 37°C. Then, the 96-well plate was centrifuged for 4 min at 300 × g to pellet the unlysed red blood cells, and supernatants were transferred to a replica 96-well plate. Hemoglobin release was measured by recording the absorbance at 420 nm of the supernatants (Spectramax i3x Plate Reader, Molecular Devices), and percent hemolysis was determined relative to red blood cells treated with positive control Triton X-100 (0.1%, Sigma-Aldrich) and negative control PBS only.

In vitro activity assays were performed as previously described for diguanylate cyclases (Burns et al., 2014 (link)) as independent technical replicates (n = 3, assays used the same stock enzyme preparation in separate reaction mixtures), with the following modifications. The EnzChek pyrophosphate kit (Life Technologies) was used according to the manufacturer’s instructions except the buffer was supplemented with KCl to a final concentration of 100 mM and MgCl₂ to a final concentration of 10 mM, and the reactions were initiated with addition of ATP or GTP. Assays were performed in triplicate in Corning Costar 96 well black, clear-bottomed plates containing 1 µM protein and varying NTP concentrations (0–10 mM). Absorbance at 360 nm in each well was measured using a SpectraMax i3x plate reader (Molecular Devices) and SoftMax Pro 6.5.1 software. Subsequent analyses to determine enzymatic rates were performed using the Excel Solver package.

The protein concentration of the individual fractions from the SEC was determined using the absorbance at 280 nm on a NanoDrop 8000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) immediately following elution from the column. DPBS was used as the blank for background subtraction. This was done to detect any protein contamination in the samples and ensure proper separation of samples.
Once the fractions were combined, the protein concentration of the 1 mL EV sample fractions were determined using a Pierce micro BCA protein assay kit (Thermo Scientific, Cat. No. 23235) according to the manufacturer’s instructions. Samples were plated in technical triplicates in a 96 well plate (Corning, Cat. No. 3370). The absorbance was read at 562 nm using a SpectraMax i3x plate reader (Molecular Devices, San Jose, CA, USA). A protein standard curve was generated using BSA standards supplied with the kit and by averaging technical triplicates. DPBS was used as a blank for background subtraction. Samples were plated in triplicate and the average was used to determine the protein content of the EVs.

Nunc 384-Well Clear Polystyrene Plates (Cat# 164688 ThermoFisher) were coated with 20 μL/well of recombinant E monomers (Cat#DENV2-ENV, Native Antigen Co, Kidlington, United Kingdom) at 3 μg/mL or 20 μL/well of antibody 4G2 at 50 μg/mL overnight. The next day plates were washed once with 50 μL wash buffer (0.05% Tween-20 in PBS) and blocked with 50 μL of blocking buffer (3% nonfat milk, Cat# 20–241 Apex Bioresearch products, in PBS) at 37°C for 45 min. Blocking buffer was aspirated from wells that had received 4G2 and replaced with 20 μL of 100X concentrated reporter virus particles diluted 1:1 in blocking buffer. Wells that had received E monomers were left in blocking buffer and plates were incubated at 37°C for 45 min. Wells were washed 3 times with 50 μL of wash buffer, received 30 μL of primary antibody at 100 μg/mL, and were incubated at 37°C for 45 min. Wells were washed 6 times with 50 μL wash buffer, received 30 μL of mouse anti-human antibody (Cat# 05–4220, ThermoFisher) at 1 μg/mL, and were incubated at 37°C for 45 min. Finally, wells were washed 6 times with 50 μL wash buffer, received 30 μL of TMB (Cat# 34028 ThermoFisher), and were incubated at room temperature until a color change was apparent. The reaction was stopped with 15 μL of 1N HCl and absorbance at 450 nm was read on SpectraMax i3x plate reader (Molecular Devices, San Jose, CA)

For adipogenic transformation, MEFs were seeded at a density of 28,000 cells/cm² at day −1. At day 0, cells were subjected to an initial adipogenic cocktail containing 1 μM dexamethasone (Sigma-Aldrich Sweden AB), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Sweden AB), 10 μg/ml bovine insulin in HEPES buffer (Sigma-Aldrich Sweden AB), and 16 μg/ml rosiglitazone (Sigma-Aldrich, Sweden AB). At day 2, the medium was thereafter changed to a cocktail containing 10 μg/ml insulin and 16 μg/ml rosiglitazone and was changed every two days until day 9 when the cells were fixed with 4% formaldehyde (Unimedic Pharma AB, Stockholm, Sweden) for 30 min and stained using a 60% isopropanol solution with 0.5% Oil Red O (Sigma-Aldrich, Sweden AB). In some experiments, 100 ng/ml recombinant murine noggin (PeproTech Nordic, Stockholm, Sweden, catalog # 250-38) was added at day −1 or 50 ng/ml recombinant human BMP4 (PeproTech Nordic, catalog # 120-05ET) was added at day 0. The Oil Red O stained cells were quantified in a Spectramax i3x plate reader (Molecular Devices, San Jose, CA, USA) using the Softmax Pro 7 software (Molecular Devices).