Amicon spin column

Transduction efficiency Ad-GFP was assessed by the presence of the fluorescent GFP protein in cells using a fluorescence Axio Observer D1 microscope with an AxioCam HRc camera (Carl Zeiss Microscopy, GmbH, Oberkochen, Germany) and by flow cytometry on a CyFlow ML (Partec, Tsuchiura, Japan).
Transduction efficiency Ad-BMP2 was assessed by BMP2 gene expression by RT-PCR and BMP-2 protein production by enzyme-linked immunosorbent assay (ELISA).
RT-PCR was carried out in a BioRad iQ cycler thermal cycler (BioRad, Hercules, CA, USA) using SYBR Green I intercalating dye (Eurogen, Moscow, Russia). Total RNA was extracted from the cells using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany) and reverse transcribed into cDNA using the RevertAid Kit (Thermo Scientific, Bremen, Germany). The expression levels of the analyzed gene were normalized to the average values of the reference genes Gapdh and Actβ.
To evaluate the production of the BMP-2 protein by MSCs, the culture medium was collected every 3 days and stored at −80 °C. Then, all fractions were combined and concentrated using a 3 kDa Amicon spin column (Merck KGaA, Darmstadt, Germany). The protein was analyzed by ELISA using a Quantikine Elisa kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocol. The measurements were performed on an xMark plate spectrophotometer.

Microscale thermophoresis (MST) was performed with the Monolith NT.LabelFree instrument (NanoTemper Technologies, Munich, Germany) [24 (link),25 (link)]. To remove residual metal ions, purified HRDs were incubated with 5 mM EDTA for 30 min before buffer exchange into Chelex (Biorad, Gladesville, NSW, Australia) treated Milli-Q water using a 3 kDa cutoff Amicon spin column (Merck, Darmstadt, Germany). Protein stocks were prepared at 40 µM (SlD26) or 80 µM (AtD90) in the appropriate MST buffer; either 20 mM HEPES, 150 mM NaCl, pH 7.4 for NiCl₂ assays, or 20 mM bis-Tris, 150 mM NaCl, pH 7.4 for all other metal salts. A fifteen step, two-fold dilution series of each metal salt (and a no metal control) in the respective MST buffer with a top concentration of 20 mM was also prepared. Protein and metal salt solutions were combined in a 1:1 ratio (10 µL of each) in a 0.2 mL tube and mixed by gentle pipetting before incubation for 30 min at room temperature. The samples were then loaded into Monolith NT Standard Treated capillaries (NanoTemper Technologies) via capillary action. Thermophoresis was monitored by intrinsic fluorescence at 25 °C with experiments performed at 20% LED power and 40% MST IR laser power. Data from three independent experiments were analyzed using the signal from Thermophoresis + T-jump employing the NT.Analysis software version 1.5.41 (NanoTemper Technologies).