106 Chapter 4 metabolic analysis to further elucidate macrophage responses to distinct collagen type I morphologies and to unravel possible molecular mechanisms by which the altered ECM in fibrosis affects macrophage function. MATERIALS AND METHODS Substrate preparation Sterile cell culture plates were coated with 75 µl/cm2 rat-tail collagen type I (Ibidi, Martinsried, Germany) at a concentration of 2.8 mg/mL at pH 3 (globular structured collagen layers) or pH 7 (fibrous collagen layers), as described before [11]. The desired pH was achieved by diluting the collagen type I stock in either 17.5 mM NaOH or 17.5 mM CH3COOH. Collagen layers were incubated at 37 °C for one hour, subsequently washed twice with sterile water and allowed to dry overnight at 37 °C. Cell culture Alveolar macrophages derived from fetal monocytes according to the protocol of Fejer et al. [12] (a kind gift by Dr. G. Fejer) were cultured in RPMI (Gibco Laboratories, Grand Island, USA) supplemented with 10% heat-inactivated FCS (Biowest, Nuaillè, France), 10 µg/mL gentamycin (Gibco Laboratories) and 20 ng/ mL murine GM-CSF (Peprotech, Rocky Hill, USA). Once a week, macrophages were detached with 1 mM EDTA (Merck, Darmstadt, Germany) and reseeded at a density of 1×105 cells/mL. The cells were incubated at 37 °C and 5% CO 2, and cell culture medium was refreshed after 4 days. Macrophages between passage number 6 and 12 were seeded at a density of 5.7×104 cells/cm2 for proteomic analysis or at a density of 35×104 cells/cm2 for extracellular flux analysis. Sample preparation for proteomic analysis Macrophages were cultured on collagen-coated or non-coated surfaces for 72 hours before harvesting for proteomic analysis. Cell culture medium was collected and centrifuged to collect detached macrophages. The cell pellet was subsequently washed in PBS. Adherent macrophages were washed five times in PBS, followed by cell lysis with SDC lysis buffer: 1% w/v sodium deoxycholate (Sigma-Aldrich, Zwijndrecht, the Netherlands) and 1 mM EDTA in 0.1 M triethylammonium bicarbonate (TEAB) buffer (Thermo Fisher Scientific, Landsmeer, the Netherlands). The lysate of the adherent macrophages was then added to the pellet of detached macrophages and mixed. Subsequently, the total lysate was denatured at 98 °C for 5 minutes and sonicated with a tip sonicator (Vibra-Cell, Sonics, Newton, United States) for 3×10 seconds, 50% without pulse. Protein concentrations were
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