166 Chapter 5 cell survival [52,53]. Samples were stored overnight at 4 °C and processed the following morning. Subject and sample characteristics are shown in Supplementary Table 1. Single cell isolation This protocol was based on the immune cell isolation procedure from He et al. [54]. Biopsies were taken from viable areas of the burn tissues, i.e. white or red areas with bleeding spots and not blackened or leathery areas. Approximately 600 mg of tissue was used per cell isolation for flow cytometry (FCM). Tissue samples were cut into smaller pieces and subsequently divided over 2 C-tubes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) containing 5 mL of RPMI 1640 containing 1% penicillin and streptomycin. C-tubes were placed on a tissue dissociator (gentleMACS, Miltenyi Biotec) and program “B” was run. Hundred-fifty μL of 80 mg/mL collagenase I (Merck, St. Louis, MO, USA) in PBS (Gibco) was added and the sample was incubated for 1 h in a shaking water bath at 37 °C. After incubation, the C-tube was placed on the tissue dissociator to run program “B”. Samples were passed through a 500 μm and 40 μm cell strainer (pluriSelect, Leipzig, Germany) to obtain a single cell suspension. Suspensions were centrifuged for 10 min at 450 × g, and supernatant was discarded. The cell pellet was resuspended in erythrocyte lysis buffer (1.5 mM NH4Cl, 0.1 mM NaHCO3 and 0.01 mM EDTA in demineralized water) for 10 min at room temperature. Twenty mL of FCM buffer (PBS containing 1% BSA, 0.05% natrium-azide and 1 mM EDTA) was added and the suspension was centrifuged for 10 min at 450 × g. The pellet was resuspended in 5 mL of FCM buffer and cells were counted on the flow cytometer (MACS Quant Analyzer 10, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Supervised flow cytometry From the single cells suspensions approximately 2.5 × 105 cells were used per staining panel. Antibodies used for FCM are displayed in Supplementary Table 2. A solution of 7-AAD (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or propidium iodide (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were used to calculate viability of cells. Stained cell samples were acquired on the MACS Quant Analyzer 10 and analysis was performed using FlowLogic (Inivai Technologies, Victoria, Australia). Gating strategy is shown in Supplementary Figure 1. Data was visualized using Graphpad version 5.01 (PRISM, La Jolla, USA) and R (ggplot package). Unsupervised flow cytometry analysis Lymphocyte (panel 1), T cell (panel 2), neutrophil (panel 3) or monocytic (panel 4) populations were gated based on FSC/SSC, CD45, CD3, CD15 in MACSQuantify 2.13.3 software (Miltenyi Biotec). Data of these sole populations were uploaded to Cytobank [55] to create Flow Self-Organizing Map (FlowSOM) clusters.
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