Deidentified blood was obtained from healthy donors [University of New Mexico (UNM) Hospital Blood and Tissue Bank and Mini Donor Dienst UMC Utrecht], and PBMCs were isolated by Ficoll-Paque gradient separation (GE Healthcare). PBMCs were allowed to rest for 1 hour in RPMI 1640–1% FCS at 37°C in (uncoated) eight-well Lab-Tek chambers. Next, PBMCs were stimulated with human TNFα and IFNγ (500 and 400 U/ml, respectively) for 1 hour in RPMI 1640–1% FCS at 37°C. During the last 10 min of incubation, IV.3 antigen binding fragments [F(ab′)2] (5 μg/ml) and 3G8 F(ab′)2 (1 μg/ml) were added to block the other FcγR (40). Wells were washed with RPMI 1640–1% FCS to remove all unbound cells, leaving the adherent cells (monocytes) in the wells. Next, monocytes were stained for SPT or super-resolution imaging.

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