Human interferon gamma

U-251 cells were grown in high glucose DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, 1% glutamine and 1% HEPES. Cells were plated at low density and allowed to adhere for 24 h, after which cells were pretreated with 0.1 μg/mL human interferon gamma (Pepro Tech Inc.) alone or in conjunction with the Lmp7 inhibitor ONX 0914 (Cayman Chemicals) at a final concentration of 0.2 μM. Following incubation overnight, 5 μg/mL α-synuclein fibrils were added to the media and incubated for 4d. The cells were washed 3× with PBS and lysed in PBS + 1% Trition-X100. 12 μg of protein was loaded into each lane and the western proceeded as described above. Primary antibodies to α-synuclein (clone Syn211, SigmaAldrich, S5566 RRID:AB_261518), Lmp7 (proteasome subunit beta type-8; Abcam, ab3329), and Actin (Sigma Aldrich, A2066 RRID:AB_476693) were incubated with the membrane at a concentration of 1:1000 overnight at 4 °C (Syn211) or for 1 h at room temp (Lmp7, Actin). IR Dye conjugated anti-mouse or anti-rabbit secondary was incubated at 1:5000 for 1 h and the blots were visualized using an Odyssey LC scanner (LiCor).

At 36–38 hours after initial culture of hUC‐MSCs, PrimeGenUS Inc. triple activation solution (Santa Ana, CA; proprietary triple activation solution by PrimegenUS and supported by exclusive licensed technology patent family: US 8,685,728; US 9,301,979; US 10,046,011; US 10,898,523; US20210213067) consisting of human TNF‐a, human interferon gamma, and IL‐17 (all from PeproTech, Inc.) was added to each T225 flask with hUC‐MSCs at final concentration of 2 ng/mL for each cytokine.^[22] Each hUC‐MSC flask as allowed to culture with added activation media for an additional 10–12 hours in at 37°C with 5% CO₂ incubator.