L glutamine solution

Experiments were performed with two immortalized human airway epithelial cell lines (16HBE14o^-, S9) and one alveolar cancer cell line (A549). With permission of D.C. Gruenert 16HBE14o^- cells were received from K. Kunzelmann (University of Regensburg, Germany). S9 cells were purchased from ATCC-LGC Standards (Wesel, Germany, S9). A549 cells were obtained from the cell collection of the Friedrich Loeffler-Institute (Riems, Germany).
Cells were cultured on 10 cm Cell+ dishes (3902300, Sarstedt, Numbrecht, Germany) in Eagle’s minimal essential medium (MEM) (P03-2950, PAN-Biotech, Aidenbach, Germany) containing 10% FBS superior (S0615, Merck, Darmstadt, Germany), 29.8 mmol/L NaHCO₃ and 1% (w/v) penicillin/streptomycin solution (P06-07050, PAN-Biotech, Aidenbach, Germany) at 37 °C and gassed with 5% CO₂. Cell culture medium of A549 cells additionally contained 1% L-glutamine solution (P04-80100, PAN-Biotech, Aidenbach, Germany). Cell culture medium was changed every 3 days. Just before cells formed confluent monolayers, they were passaged onto new cell culture dishes or used directly for experiments. All cell cultures were checked for Mycoplasma contaminations on a regular basis.

The murine melanoma cell line B16.F10 was cultivated in Dulbecco's modified Eagle's medium (DMEM) with the addition of 10% (V/V) fetal bovine serum (both from Sigma‐Aldrich Chemie GmbH), 1% (V/V) penicillin/streptomycin solution and 1% (V/V) l‐glutamine solution (both from PAN Biotech GmbH, Aidenbach, Germany) as described previously 11. HEK293 cells were cultured in DMEM supplemented with 10% (V/V) fetal bovine serum, 1% (V/V) penicillin/streptomycin at 37 °C and 5% CO₂ (Sigma‐Aldrich Chemie GmbH).
Tests for the absence of mycoplasms were performed routinely every month. LysoPC treatment of B16.F10 cells was performed with LysoPC C18:0 or LysoPC C18:1, achieving a final concentration of 450 μm and 2% (m/V) BSA for an incubation time of 72 h.
To prepare lysates of the LysoPC‐treated and untreated B16.F10 cells, cells in sub‐confluent flasks were washed twice with ice‐cold PBS (PAN Biotech GmbH) and incubated with lysis‐buffer and 1 mL of cell extraction buffer (both from Life Technologies GmbH, Darmstadt, Germany) for 10 min. Afterwards, cells were manually detached from the cell culture flasks and the cytosolic fraction was isolated via centrifugation at 17 000 g for 15 min.