Myiq2 real time pcr instrument

Thermal shift assays were performed using a Bio-Rad MyIQ2 Real-Time PCR instrument. Assay mixtures (25 μl) contained 20 μM protein, SYPRO^® Orange (Life Technologies) at 5× concentration and ligands at final concentrations of 0.5–2 mM. Samples were heated from 23 to 85°C at a rate of 1°C min⁻¹. The protein unfolding curves were monitored by detecting changes in SYPRO^® Orange fluorescence.

Thermal shift assays were performed on a MyIQ2 Real-Time PCR instrument (BioRad). Ligands from the different compound arrays (Biolog, Hayward, CA, USA; see Supplementary Fig. S3) were dissolved in 50 μl of MilliQ water, which, according to the manufacturer, corresponds to a concentration of 10–20 mM. Screening was performed using 96 wells plates. Each well contained 20 μM of protein dialyzed into TNG buffer (20 mM Tris/HCl, 150 mM NaCl, 10% (v/v) glycerol, pH 6.7), 2.5 μl of the resuspended compounds and SYPRO Orange (Life Technologies) at 5× concentration. In a single well (ligand free protein) the compound was substituted by water. Samples were heated from 23 °C to 85 °C at a scan rate of 1 °C/min. The protein unfolding curves were obtained by following the changes in SYPRO Orange fluorescence. Melting temperatures were determined using the first derivative values from the raw fluorescence data.

Using DSF, changes in the midpoint of protein unfolding transition (Tm) of a protein can be recorded. Typically, ligand binding stabilizes the protein and the identification of compounds that cause an increase in the Tm value is an evidence for ligand binding (Martin‐Mora et al., 2018a ). DSF assays were performed using a Bio‐Rad MyiQ2 Real‐Time PCR instrument. Ligands from different compound arrays (Biolog, Hayward, CA, USA; for further information, refer to http://www.biolog.com/) were dissolved in 50 μl of Milli‐Q water, which, according to the manufacturer, corresponds to a concentration of 10–20 mM. Assay mixtures (25 μl) contained 20–50 μM protein dialyzed in buffer C [50 mM Tris, 150 mM NaCl, 5% (vol/vol) glycerol, pH 8.0; PcpI‐LBD] or buffer D [5 mM Tris, 5 mM Pipes, 5 mM Mes, 10% glycerol (vol/vol), 150 mM NaCl, pH 8; E6B08_RS28125], SYPRO® Orange (Life Technologies) at 5× concentration and ligands at final concentrations of 1–2 mM. Samples were heated from 23°C to 85°C at a rate of 1°C min⁻¹. The protein unfolding curves were obtained by monitoring the changes in SYPRO Orange fluorescence. Tm values correspond to the minima of the first derivatives of the raw fluorescence data.

Fluorescence-based thermal shift (FTS) assays were performed using a BioRad MyIQ2 Real-Time PCR instrument. Each 25 μl assay mixture contained 1 μM FleQ and SYPRO Orange at 5× concentration in STAD [25 mM Tris-acetate pH 8.0, 8 mM Mg-acetate, 10 mM KCl, 3.5% (w/v) polyethylene glycol-8000 and 1 mM DTT]. The nucleotides were prepared as 10× stocks and aliquots of 2.5 μl were added to each well. Samples were heat denatured from 20 to 90°C at a ramp rate of 0.5°C/min. The protein unfolding curves, both in the absence and in the presence of the nucleotides, were monitored by detecting changes in SYPRO Orange fluorescence and the first derivative values (-dF/dt) from the raw fluorescence data were used to determine the melting temperature (Tm). All experiments were performed in triplicate.

Thermal shift assays were performed using a BioRad MyIQ2 Real-Time PCR instrument. Ligands were prepared by dissolving Biolog Phenotype Microarray compounds in 50 μl of MilliQ water to obtain a final concentration of around 10–20 mM (as indicated by the manufacturer). Screening was performed with plates PM1, PM2A, PM3B, PM4A, and PM5. Each plate contains 95 compounds and a control (Supplementary Figure S1). Each 25 μl assay mixture contained 40 μM protein in 5 mM Tris, 5 mM Pipes, 5 mM Mes, pH8.0 and SYPRO orange (Life Technologies) at 5x concentration. Aliquots of 2.5 μl of the resuspended Biolog compounds were added to each well. Samples were heated from 23°C to 85°C at a scan rate of 1°C/min. The protein unfolding curves were monitored by detecting changes in SYPRO orange fluorescence. Melting temperatures were determined using the first derivative values from the raw fluorescence data.

The expression of chemokine, cytokine, and neurotrophin genes, as well as those associated with phagocytosis, was determined first with RT² Profiler PCR arrays (Qiagen) on a Bio-Rad myIQ2 Real-Time PCR instrument. The manufacturer’s software was used to analyze the data. Alterations in individual genes were confirmed using individual PCR primer sets for qRT-PCR (Qiagen; Table 1). Individual reactions contained 5.5 μl nuclease-free water, 2 μl primer mix (both forward and reverse) at 10 μM, and 12.5 μl Bio-Rad Sybr Green Supermix ±5 μl template DNA or no template control (nuclease-free water). Forty cycles of PCTR were performed following an initial 10 min denaturation at 95°C. For each cycle, a 1 min annealing step at 60°C preceded a 15-s melting interval at 95°C. Melting curves were obtained using a stepped temperature gradient of 0.5°C × 10 s starting at 60°C. Transcript expression levels were then compared to standard housekeeping genes (β-actin and GAPDH) and to those of untreated cells using the 2^-ΔΔCT method, where C_T (threshold cycle). This method was used on each individual example with the untreated sample as the comparator (Schmittgen and Livak, 2008 (link)). Triplicate samples were analyzed from a minimum of three independent biological replicates for each time point.