Kaluza analysis 1.5a

MSC were trypsinized from the culture flasks at 90% confluency or thawed after cryopreservation and re-suspended either in complete culture medium or perfusion fluid at a concentration of 500,000 MSC/ml. MSC were incubated in perfusion fluid in polypropylene tubes to avoid attachment of MSC to plastic. After 30 min or 2 h in suspension, MSC were submitted to a RBC lysis process to remove the large amount of RBC present in perfusion fluid. MSC incubated in suspension with culture medium were also subjected to RBC lysis to treat both groups in the same way. Briefly, 3 ml of red blood cell lysis buffer (Invitrogen, Carlsbad, CA, USA) was added to MSC and incubated for 20 min at room temperature (RT). MSC were then washed with PBS and stained with Annexin-V (PE) and ViaProbe (PercP) to assess the number of early and late apoptotic cells. Perfusion fluid was also added to attached MSC and incubated for the same time and then trypsinized and stained as mentioned. Cells were analyzed by flow cytometry (FACS Canto II, BD Biosciences, NJ, USA) and data were analyzed using Kaluza Analysis 1.5a (Beckman Coulter, Brea, CA, USA).

Cells were harvested and washed twice with cold phosphate-buffered saline (PBS). To analyze the percentage of nuclei with hypodiploid content (Sub-G1), nuclei were stained by using PI and at least 20,000 events were analyzed by a FACScan and Kaluza Analysis 1.5a (Beckman Coulter).

The fluorescein isothiocyanate FITC Annexin V Apoptosis Detection Kit I (FITC Annexin V Apoptosis Detection Kit I, BD Bioscences, NJ, USA) was used to assess apoptotic cell death. Assays were prepared in 12-well plates by adding 1 × 10⁶ cells/well and after 24 h test compounds were added at the appropriate concentrations and incubated for 48 h. The positive control comprised cells treated for 16 h with 1 μM staurosporine (Sigma-Aldrich Corp., St. Louis, MO, USA), while negative control represented cells incubated for 48 h in complete culture medium. Afterwards, the cells were washed twice with cold phosphate-buffered saline (PBS) (Sigma-Aldrich Corp., St. Louis, MO, USA) and then double-stained with: annexin V (an early apoptosis marker) and propidium iodide (PI) (cell membrane disintegration, necrosis, and late apoptosis marker). CytoFLEX (Beckman Coulter, Brea, CA, USA) was used to calculate the percentage of apoptotic cells by flow cytometry (FC) and the obtained data were analysed using Kaluza Analysis 1.5 A (Beckman Coulter, Brea, CA, USA)^{30 (link),31 (link)}.