Macrophage Polarization and Mitochondrial Function

Bone marrow derived macrophages (BMDM) were differentiated for 5 days in RPMI 1640 media with 15% fetal calf serum (FCS) and antibiotic-antimycotic solution supplemented with M-CSF (20 ng/ml). Polarization of BMDM was performed for additional 3 days RPMI 1640 media with 15% fetal calf serum (FCS) with 100 ng/ml LPS (Sigma–Aldrich, St. Louis, MO, USA) and 10 ng/ml IFNγ (PeproTech, Rocky Hill, NJ) for M1 macrophages and with IL-4 (10 ng/ml, PeproTech, Rocky Hill, NJ) for M2 macrophages. CO treatment was applied at 250 ppm in CO chamber (5% CO₂, 21% O₂ and 95% humidity) as previously described [18 (link)].
PC3 cells were co-cultured with BMDM from Hmox1^fl/fl or LyzM-Cre:Hmox1^fl/fl mice in a 1:2 or 1:5 ratio for 24 h. PC3/BMDM cells alone or in co-culture were used for immunostaining using anti-E-cadherin antibody (CatNo: ab15148) and Mitotracker Red CMXRos (Life Technologies), for detecting mitochondria as well as for direct testing of mitochondrial activity Mitochondria Stress Kit (Seahorse) was used as previously described [49 (link)].
Overexpression of HO-1: Constructs were kindly provided by Dr. Chau (Taiwan) and forced overexpressions of Flag, HO-1-Flag or truncated HO-1-Flag (t-HO-1) were established in PC3 cells upon transfection with Amaxa. Forty eight hours after transfection, cells were stained or cell lysates were immunoblotted.