Fig. 2.

Survival of the MSCs under the culture conditions, comparisons of four donors, and demonstration that most of the secreted proteins and EVs are anionic. (A) Survival of MSCs in CDPF. MSCs were expanded to about 70% confluence and then incubated an additional 48 h in CCM, CDPF, or PBS. (Top) Cultures labeled with Hoechst, Calcein AM, and propidium iodide (PI) demonstrate viable cells in CDPF but not PBS. (Bottom) Assays of the same cultures by flow cytometry after labeling with PI and Annexin V demonstrated survival in CCM or CDPF medium but not PBS. (B) Comparisons of four different preparations of MSCs (donors 235, 6015, 7052 and 7074) after incubation as in Fig. 1G. (i) CD63⁺ in medium assayed by ELISA. (ii) TSG-6 in medium assayed by ELISA. (C) Small-scale assays in SDS-electrophoretic gels demonstrated that most of the medium proteins bound to and were eluted from an anionic resin and not a cationic resin. Gels were stained with silver.