2.4. Preparation of Fractions of MSC Conditioned Medium

MSC were grown in T175 flasks until reaching 80% confluency. MSC were rinsed in PBS and fresh StemSpan SFEM II medium was added. Afterwards, MSC were 2 Gy irradiated by 6 MV X-rays in a Versa HD linear accelerator (Elekta, Stockholm, Sweden), while control MSC were not irradiated. MSC conditioned medium and control medium were obtained from irradiated and non-irradiated MSC, respectively, after 4 h incubation at 37 °C. The collected medium was centrifuged (4000× g, 10 min) and supernatants were filtered through 10 kDa molecular weight cut-off (MWCO) ultrafiltration centrifugal filter units (Amicon Ultra, Merck, Darmstadt, Germany) to obtain (I) approximate <10 kDa fractions of MSC conditioned and control medium, respectively. Next, the supernatants above the filter were adjusted with fresh medium to the original volume and filtered through 100 kDa MWCO ultrafiltration centrifugal filter units to obtain (II) approximate 10–100 kDa fractions of MSC conditioned and control medium, respectively. Finally, the supernatants above the filter were adjusted with fresh medium to the original volume and then contained (III) approximate >100 kDa fractions of MSC conditioned and control medium, respectively. The distinct fractions (I)–(III) of MSC conditioned and control medium were stored at −20 °C.