Cfx384 touch machine

For qPCR analysis, protein were added to plated cells and incubated for either 2 or 8 h at 37 °C. Cells were then collected and RNA isolated as described below. Total RNA was isolated from treated BV2 cells using TRIzol reagent (LifeTechnologies), following the manufacturer protocol. Reverse transcription of 200 µg total RNA was performed using SuperScript III reverse transcriptase (LifeTechnologies) and random hexamer primers (LifeTechnologies). Semi-quantitative RT-PCR was performed using 1 µL of the resulting cDNA in a 5 µL total volume containing SSoAdvanced Sybr Green (BioRad) and murine primers (IDT) targeting the genes of interest. For amplification and recording, a CFX384 Touch machine (BioRad) was used, and the results were evaluated using the manufacturer’s software. Amplification specificity was confirmed by melting curve analysis, with quantification performed using the ΔΔCt method.

Recombinant IgG and scFv-Fcs were expressed in Expi293 cells (ThermoFisher, Waltham, MA, USA) and purified using protein A Fast protein liquid chromatography with subsequent preparative size exclusion chromatography (SEC). Proteins were subsequently exchanged into a phosphate-buffered saline (PBS) and characterized using SDS-PAGE, Western blotting, analytical SEC, and the SYPRO orange ThermoFleur assay.
Proteins were characterized as a function of temperature using the ThermoFleur assay through the addition of 5-μM protein to SYPRO orange. Temperature and fluorescence monitoring were performed using a CFX384 Touch machine (Bio-Rad, Hercules, CA, USA), with temperature increased from 25 °C to 90 °C; samples were incubated for 5 s at each temperature prior to fluorescence measurement [24 (link),25 (link)].