3 kda ultrafiltration centrifuge tubes

The following six TSC CM were prepared: CM1, LG-DMEM only; CM2, LG-DMEM with TSCs; CM3, LG-DMEM with 10 ng/ml HGF (PeproTech) + TSCs; CM4, LG-DMEM with 20 ng/ml HGF + TSCs; CM5, LG-DMEM with 40 ng/ml HGF + TSCs; and CM6, LG-DMEM with 80 ng/ml HGF + TSCs. The same number of TSCs were used in each preparation. After 24 h of culture, the supernatant from CM2–6 was collected and centrifuged, and the new supernatant was filtered to remove cell debris. The filtrate was divided into 2 equal parts. One portion was used in in vitro experiments and the other was concentrated using 3-kDa ultrafiltration centrifuge tubes (Millipore, Billerica, MA, United States) at 6000 × g for 1 h at 4°C for in vivo studies.

APT solutions were filtered through 3 kDa ultrafiltration centrifuge tubes (Millipore, MA, USA). After desalination, the components whose M_w were under 3 kDa were subjected to sequence identification by Q Exactive HF-X (Thermo Scientific, Waltham, MA, USA) [27 (link)]. The peptide sequences were obtained by PEAKS Studio software (Bioinformatics Solutions Inc., Waterloo, ON, Canada).