PMA/ionomycin T cell activation.

Splenocytes and SVF were purified from spleen and eWAT and were resuspended in a single-cell suspension. Cells were plated at 4 × 10⁵ cells/well in a 96-well round-bottom plate and rested overnight. The next morning, 10 μg/mL of Brefeldin A was added to cultures to block protein secretion from the Golgi apparatus. After 1 hour, SVF was stimulated with 10 ng/mL of PMA and 250 ng/mL of ionomycin. After stimulation for 4 hours, cells were collected, washed, and stained for flow cytometry to assess live ATT subpopulations and intracellular IFN-γ (Alexa Fluor 647 IFN-γ [catalog 505814] from BioLegend).