Facs melody device

The human squamous cell carcinoma cell lines FaDu (hypopharynx), Cal33 (oral tongue), XF354 (oral cavity) and SAS (oral tongue) were a kind gift of Prof. Daniel Zips and have been cultured in RPMI-1640 medium supplemented with 10% fetal bolvine serum (FBS), 100 Units/ml penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham), 2 mM L-Glutamine and 1 mM sodium pyruvate (Sigma Life Science, St. Louis) at 37 °C and 5% CO₂. SAS subclones were randomly isolated (no selection/surface marker) via single cell sorting into 96 well plates filled with culture medium containing 2.5 μg/ml amphotericin B using a FACS Melody device (BD Bioscience, New Jersey). A total of twenty subclones were further cultivated and used in subsequent experiments. For chemical LCK inhibition, cells were treated with dasatinib (Sigma-Aldrich, St. Louis) or A-770041 (Axon Medchem, Reston) as indicated.

Cultured neutrophils were labeled with FVS780 viability stain (BD Biosciences, San Diego, CA, USA) and Sytox Green (Thermo Fisher Scientific). Fluorescence was detected using a FACS Melody device (BD Biosciences, San Jose, CA, USA). Analysis was performed using the FlowJo software (Becton Dickinson and Company, Franklin Lakes, NJ, USA).