L glutamine

Human primary leukocytes were isolated from peripheral blood of healthy adult volunteers provided by the Institute of Transfusion Medicine at the University Hospital Greifswald as described before [45 (link)]. All methods were performed in accordance with the Declaration of Helsinki. In brief, erythrocytes were removed by dextran sedimentation and leukocytes were separated by the density gradient centrifugation on a lymphocyte separation medium (Histopaque^®-1077, Merck, Darmstadt, Germany). The remaining erythrocytes were removed by hypotonic lysis using water, and resulting polymorphonuclear neutrophils (PMNs) were resuspended in PBS containing 0.1% (w/v) glucose (PG buffer) or PG buffer with 1 mM CaCl₂ (PGC buffer) as indicated. Resulting peripheral blood mononuclear cells (PBMC), including monocytes, were seeded in the RPMI 1640 medium supplemented with 10% (v/v) FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1.5 h at 37 °C and 5% CO₂. Adherent monocytes were washed twice with PBS and were finally resuspended in the RPMI 1640 medium as described before [45 (link)].

BMMC and BMMØ were cultured as previously described in (5 (link)). Briefly, bone marrow was isolated from femurs of wild-type and transgenic mice. To initiate differentiation of BMMCs, bone marrow cells were cultured in IMDM supplemented with 10% heat-inactivated fetal calf serum (HI-FCS), 2 mM L-Glutamine (Sigma, G7513), 100 U/ml Penicillin + 100 µg/ml Streptomycin (Sigma, N109), 50 μM β-mercaptoethanol (Sigma M6250) and 2 ng/ml murine interleukin-3 (IL-3, Peprotech 213-13). Recombinant murine SCF (5 ng/ml, Peprotech 250-03) was added only during the first passage. c-kit⁺FcεRI⁺ BMMCs were used for experiments after 4 weeks of culture. Culture medium was supplemented with IL-3 (2 ng/ml) every two days.
To generate BMMØ, bone marrow cells were cultured in bacterial petri dishes (Greiner bio-one 633180) in RPMI supplemented with 10% HI-FCS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin, 50 μM β-mercaptoethanol and 20% L929-conditioned medium. Non-adherent cells were collected after 5 days of culture to perform experiments.
For BMMC and BMMØ GPCR activation adenosine (Fluka Bio Chemika 01890), recombinant mouse C5a (R&D Systems #8085-C3-025) and platelet activating factor (PAF; Sigma, P7568) were used.