8 well glass based imaging dish

Human macrophages were derived from monocytes isolated from the blood of healthy volunteers. PBMCs were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Slough, UK) supplemented with 200 U/ml penicillin/streptomycin antibiotics (Invitrogen, Paisley, UK) and 2 mM l-glutamine (Invitrogen, Paisley, UK). Serum isolated from blood was heat inactivated for 20 min at 56 °C. PBMCs were seeded at 6 × 10⁵ in 300 µl/well supplemented DMEM containing 10% autologous human serum, onto an 8-well glass-based imaging dish (Ibidi, Munich, Germany) and incubated at 37 °C with 5% CO₂ for 1 h 45 min to facilitate monocyte adherence to the glass surface. Floating lymphocytes in the supernatant were aspirated and the same volume of fresh pre-warmed supplemented DMEM containing 10% autologous human serum added to the well. Cells were incubated at 37 °C, 5% CO₂ for 7 days with media changed on days 3 and 6^{64 (link)}. Cells were used in imaging experiments on day 7. Supplemented DMEM was replaced with pre-warmed supplemented CO₂-independent media containing 1 µM LysoTracker Red DND-99 (Invitrogen) immediately prior to phagocytosis experiments.

J774.1 macrophages (ECACC, HPA, Salisbury, UK) were maintained in tissue culture flasks in DMEM (Lonza) supplemented with 10% (v/v) FCS (Biosera, Ringmer, UK), 200 U/ml penicillin/streptomycin antibiotics (Invitrogen) and 2 mM l-glutamine (Invitrogen) and incubated at 37 °C, 5% CO₂. For phagocytosis assays, macrophages were seeded in supplemented DMEM at a density of 1 × 10⁵ cells/well in an 8-well glass-based imaging dish (Ibidi) and incubated overnight at 37 °C, 5% CO₂. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with pre-warmed supplemented CO₂-independent media (Gibco) containing 1 µM LysoTracker Red DND-99 (Invitrogen).