X vivo 10 hematopoietic cell medium

Peritoneal macrophages were harvested by lavage from mice of both sex injected with 2mL intraperitoneal (IP) administration of 4% thioglycolate (Sigma, Cat# T9032) 4 days prior to collection (Febbraio et al., 2000 (link)). Collected cells were counted, centrifuged at 250g for 5min, resuspended in RPMI media (Gibco by Life Technologies) supplemented with 10% FBS, 100U/mL penicillin and 100 μg/mL streptomycin (Sigma) and seeded into culture dishes or plates for further analysis. Cells were maintained at 37°C in a humidified incubator set at 5% CO2. Human monocyte-derived macrophages (HMDMs) were generated from monocytes isolated from human buffy coats by Ficoll density centrifugation and then differentiated over 7 days in vitro. (Chen et al., 2017 (link); Johnson et al., 1977 (link)). HMDMs were maintained in X-Vivo 10 hematopoietic cell medium (Lonza, Cat# 04-380Q) containing 5% human serum (Sigma, Cat# H3667) and 1% penicillin/streptomycin. The cells were serum-starved overnight and then treated with LPS diluted in serum-free medium. LPS was purified from Escherichia coli Gram-negative bacteria consisting of a lipid A moiety linked to an antigenic O-polysaccharide (Smooth-form) (Sigma, Cat#L2880). The THP-1 Dual cell line was a kind gift from Dr. Kathryn J. Moore (New York University).