Fitc anti human cd14 antibody

Buffy coat was obtained via the blood bank of Deutsches Rotes Kreuz (Springe, Germany) from pooled samples of voluntary, anonymous blood donors who gave informed consent. Lymphocytes were prepared from buffy coat by Ficoll-Hypaque density gradient centrifugation⁵⁷ . Buffy coat was diluted in PBS (ratio 1:3) and Ficoll-Hypaque was added, following centrifugation for 20 min at 800×g. Afterwards, 5 × 10⁸ cells were cultured in Roswell Park Memorial Institute (RPMI) medium containing 5.5 g × l⁻¹ NaCl, 5.0 mg × l⁻¹ phenol red, 2.0 g × l⁻¹ NaHCO₃, 25 mM HEPES, 4 mM stable glutamine without sodium pyruvate (Biochrom), 100 units × ml⁻¹ penicillin, 100 µg × ml⁻¹ streptomycin, 2.5 ng × ml⁻¹ GM-CSF (granulocyte-macrophage colony-stimulating factor) and supplemented with 10% human plasma. After o/n incubation at 37 °C in an atmosphere of 5% CO₂ and 90% humidity, non-adherent cells were removed, and adherent cells were cryo-preserved in inactivated fetal calf serum (iFCS) and DMSO. Next, cell suspensions were thawed, seeded, and maintained in RPMI-1640 (Biochrom), supplemented with 20% FCS and 2.5 ng × ml⁻¹ GM-CSF (Peprotech) to differentiate monocytes into macrophages. After 5–7 days, the purity of the macrophage population was checked by staining with FITC anti-human CD14 antibody (BioLegend) and FC analyses. The macrophages were used for infection by STM strains.

Human buffy coat preparations were used as a source of peripheral blood mononuclear cells (PBMCs) isolated by centrifugation over Ficoll-Histopaque (Sigma-Aldrich) density gradient. PBMCs were resuspended (10-15×10⁶/mL) in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM Hepes, 5 μg/mL gentamycin and 10² U/mL penicillin/streptomycin (all from Lonza). Highly enriched monocytes (> 85% CD14+ as verified by flow cytometry following staining with FITC anti-human CD14 antibody [BioLegend, London, UK]) were obtained from PBMCs by plastic adherence for 2 h at 37°C in a humidified 5% CO₂ incubator. Macrophages were differentiated from monocytes by incubation with 100 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) for 5 days [24 (link)]. Differentiation was confirmed by flow cytometry, by staining macrophages with APC-Cy7 anti-human CD206 antibody (BioLegend). Both monocytes and macrophages were in vitro infected with L-78 or ATCC 43816. The levels of proTα(100-109) were quantified in supernatants by ELISA, whereas the type of induced cell death and their phagocytic ability were analyzed by flow cytometry and confocal microscopy.

Monocytes were isolated from buffy coat of pooled blood samples from anonymous donors (obtained from the Deutsches Rotes Kreuz) and differentiated into macrophages as described in Bonifacino et al. (2004) , section 2/6 – 2/9. Briefly, buffy coat was mixed 1 + 1 with PBS and applied to centrifugation on a Ficoll-Hypaque gradient. A white ring containing leukozcytes including peripheral blood mononuclear cells was collected, isolated cells were quantified and cryopreserved. Next, cell suspensions were thawed, seeded and maintained in RPMI-1640 (Biochrom), supplemented with 20% FCS and 2.5 ng x ml^-1 GM-CSF (Peprotech) to differentiate monocytes into macrophages. After 5–7 days, the purity of the macrophage population was checked by staining with FITC anti-human CD14 antibody (BioLegend) and FC analyses. The macrophages were used for infection by STM strains.