Buffy coats

Murine BMM (from 8 to 12 week old C57BL/6 mice) and human MDM were prepared as before (Fleetwood et al., 2015 (link)). Briefly, bone marrow cells were isolated from femurs of mice and cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated FCS, 2 mM GlutaMax-1, 100 U/ml penicillin, and 100 μg/ml streptomycin in the presence of M-CSF (2,000 U/ml). On day 4, non-adherent cells were collected and cultured for a further 3 days in M-CSF (2,000 U/ml) to derive BMM. Adherent BMM were harvested on day 7 at which stage they express the major macrophage surface markers (e.g., CSF-1R, F4/80, and Mac-1) (Lari et al., 2007 (link)). Human monocytes were purified from buffy coats (Red Cross Blood Bank, Melbourne, VIC, Australia), using RosetteSep Ab mixture (Stem Cell Technologies, Vancouver, BC, Canada), which negatively selects CD14⁺ monocytes, followed by Ficoll-Paque density gradient centrifugation (Way et al., 2009 (link)). Cells were then cultured in RPMI 1640 (supplemented as above) for 7 days in M-CSF (2,000 U/ml) to differentiate them into MDM (Lacey et al., 2012 (link)). All experiments were approved by the Royal Melbourne Hospital Research Foundation and Animal Ethics Committee and conducted in compliance with the guidelines of the National Health and Medical Research Council.

CD14 positive monocytes were purified from buffy coats purchased from The Blood Center (New Orleans, LA), using EasySep Direct Human Monocyte Isolation Kit for immunomagnetic negative selection (STEMCELL Technologies). Cells were seeded at 2x10⁶ cells/ml in serum-free CTL test media, incubated for 3 days with or without 10ug/ml LTA1 and had supernatants were collected.