Calcium Flux Assay

Calcium flux assay was performed as previously described (Tu et al., 2018). Briefly, a sample of 1 × 10⁶ cells was resuspended in RPMI-1640 (Roswell Park Memorial Institute-1640) medium with no phenol red (Invitrogen, Grand Island, NY), 25 mM of HEPES (Invitrogen, Grand Island, NY) and labeled with Fluo-4AM (Invitrogen, Grand Island, NY) at a final concentration of 1 μM for 30 minutes, then washed and resuspended in fresh medium. Fluorescence intensity was measured for 20 seconds to establish baseline, then stimulus was added directly into the sample. Purified recombinant CXCL12 (Peprotech, Rocky Hill, NJ), cmvIL-10, Oncostatin-M, and IL-6 (R&D Systems) were used at 100 ng/ml. Sample data was collected using a BD Accuri C6 Flow Cytometer and analysis performed with FlowJo software.