Macrophage viability assay

PBMCs were isolated from whole blood from healthy controls using Histopaque and rested overnight at 37^oC 5% CO₂. CD14⁺ monocytes were then isolated by Miltenyi Biotec positive selection kit. Isolated CD14⁺ were suspended in RPMI 1640 containing 10% FBS and glutamine (R10) at 0.5x 10⁶ / ml cell density and plated out in 60 x 25mm mono plates.

Monocyte differentiation into macrophages:

1. Monocytes were cultured in R10 medium supplemented GM-CSF (20ng/ml) and cultured for 6 days at 37^o Medium (R10) + GM-CSF was re-supplemented at day 3. Cells were harvested at day 6 using 'Accutase' (1 ml per plate for 20 minutes at 37^oC, then washed with 10 ml of R10 and collected after centrifugation for 10 min at 300Xg).
2. Macrophages were plated at 2 x 10⁴ cells / well and left a further 3 days with or without the addition of compounds (1µM final concentration or DMSO control).
3. At the end of the 3 days, wells were stimulated with 20ng/ml LPS, 20ng/ml IFN-γ and left for 24 hours.
4. 6 hrs prior to the end of the assay PrestoBlue® 10X reagent was added to microplate wells. Incubate at 37°C for 6 hrs.
5. Read fluorescence at 590 nm.