Biocoll density gradient

Human peripheral blood was obtained from Etablissement Francais du Sang (EFS) after obtaining informed consent from the donor. Human peripheral blood mononuclear cells (PBMC) were isolated using a Biocoll density gradient (Biochrom GmbH, Berlin, Germany). Cells were washed in PBS 3% FCS and diluted to the appropriate concentration in 1× PBS before injection into mice.

Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a Biocoll density gradient (Biochrom, Berlin, Germany) with centrifugation at 800 g for 30 minutes at room temperature with no brake. Buffy coats from healthy volunteers were obtained with written informed consent by the German Red Cross Organization, Berlin, Germany. The use of buffy coats in the study was approved by the local ethics committee of the Charité-Universitätsmedizin Berlin as EA 1/226/14. After PBMCs were harvested from the interphase and washed three times with cold phosphate-buffered saline (PBS; Biochrom), they were either used for CD14⁺ monocyte isolation or to prepare stimulated supernatants.

PBMC were isolated from EDTA-anticoagulated blood of healthy donors using standard Biocoll density gradient centrifugation (Biochrom AG, Berlin, Germany), as approved by the Ethical Committee of the Medical Faculty, Friedrich-Alexander-Universität Erlangen-Nürnberg (Ref. no. 3299). PBMC were cultivated in RPMI 1640 with supplements described above. For generation of macrophages, PBMC were seeded into Nunc Lab-Tek chamber slides (Thermo Fisher Scientific) and cultivated in the presence of 15% heat-inactivated autologous serum, removing non-adherent cells by trypsin after 3 days. At 10–14 days, macrophages were infected with wild type HSV-1 (32 (link)), HSV-1 166v (33 (link)), HSV-1 d106S, and HSV-1 d106S-MelanA. MRC-5 fibroblasts (ATCC® CCL-171^TM) and melanoma cell lines (IGR-37, IGR-39, ARST-1, ICNI-5li, SK-MEL30, LIWE-7) were cultivated as described (9 (link)).