Percoll

Human neutrophils were separated from whole blood or buffy coats by discontinuous density-gradient centrifugation on Percoll (Biochrom) as previously described [18 (link)]. After hypotonic lysis to remove contaminating erythrocytes, cells were washed with in PBS. Purity and viability were routinely > 95% as assessed by forward and side scatter characteristics of FACSCalibur (BD Biosciences) and trypan blue exclusion, respectively.

Up to 10 ml aspirated iliac crest bone marrow were diluted 1:2 with PBS (PAA) and layered onto Percoll (density 1.124 g/ml; Biochrom, Berlin, Germany) diluted to a density of 1.068 g/ml. After centrifugation at 800g for 20 min at room temperature, mononuclear cells (MNC) were collected from the interphase, washed twice with PBS, and plated at a density of 4x10⁴/cm² in α-MEM (#E15-862, PAA) supplemented with 100 U/mL penicillin (PAA), 100 μg/mL streptomycin (PAA), 2 IU/ml heparin (Ratiopharm), and 5% freshly thawed platelet lysate [12 (link)]. Cells were incubated at 37°C and 5% CO₂. Non-adherent cells were washed off with PBS after 2–3 days. Medium was changed twice a week.
When cultures reached about 90% confluence, cells were detached with 0.05% Trypsin/0.02% EDTA (PAA), counted, and re-plated at 500 cells/cm² in 175 cm² flasks (Saarstedt). Under these culture conditions, MSC maintain multilineage differentiation capacity, express characteristic surface marker proteins (CD59, CD90, CD105), lack expression of hematopoetic markers, and remain cytogenetically stable at least until passage 6 [12 (link)].