224 Chapter 7 the transwells (Figure 6B). FSEs with monocytes were cultured for 7 more days with a medium change at day 3. T cell FSE Lymphocytes were isolated by culturing PBMCs in a culture flask. After 24 h, adherent cells were removed. T cells were activated by adding anti-CD3/CD28 Dynabeads (Gibco) at a bead/cell ratio of 5:1 at 37 °C for 4 h. After the activation, cells were resuspended in FSE I medium and 2.5 × 105 cells were placed between the transwell membrane and the dermal side of the FSE (Figure 6C), based on previous findings [35]. FSEs with T cells were cultured for 3 more days. Cell isolation from immune cell model The immune cell isolation procedure was based on a protocol from He et al. [57]. Macrophage FSEs were incubated with 0.25 U/ml collagenase A (Roche) at 37 °C in a shaking water bath for 20 min. Because enzymes affect the expression chemokine receptor [58], T cell models were not dissociated using collagenase A. FSEs were then put in C-tubes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with 5 mL of PBS containing 1 mM EDTA and further dissociated by running program “B” twice on a tissue dissociator (gentleMACS, Miltenyi Biotec GmbH). Samples were passed through a 500 µm and 40 µm cell strainer (PluriSelect) to obtain a single cell suspension. Flow cytometry Single cell suspensions were stained using the macrophage or T cell panel (Supplementary Table 2). Zombie Aqua (BioLegend, San Diego, CA, USA) was used in the macrophage panel and propidium iodide (Miltenyi Biotec GmbH) was used in the T cell panel to determine viability of cells. Stained cell samples were acquired on the flow cytometer (MACS Quant Analyzer 10, Miltenyi Biotec GmbH) and gating (Supplementary Figure 1) was performed in FlowLogic (Inivai Technologies, Victoria, Australia). Immunohistochemistry See Supplementary Table 3 for antigen retrieval and primary antibodies. Kryofix (50% ethanol + 3% PEG300 in demineralized water)-fixed paraffin-embedded samples were cut into sections with a thickness of 5 µm and rehydrated followed by hematoxylin and eosin staining or blocking of endogenous peroxidase using 1% hydrogen peroxide at room temperature for 15 min. After antigen retrieval was performed, sections were pre-incubated with 5% normal goat serum (Sigma-Aldrich) diluted in PBS + 1% bovine serum albumin (ThermoFisher). Sections were then incubated with primary antibodies at room temperature for 1 h followed by incubation with a poly-HRP-goat-anti-mouse or rabbit secondary antibody (BrightVision, VWR, Amsterdam, the Netherlands) for
RkJQdWJsaXNoZXIy MTk4NDMw