Rpmi media

For neutrophil isolation, blood was drawn from gender and age-matched HD patients before the start of a dialysis session and from HC subjects. Human neutrophils were isolated from 3 ml up to 20 ml of whole blood samples (according to the desired cell numbers per application) in EDTA blood sampling tubes. Highly pure neutrophils were isolated using the EasySep™ Direct Human Neutrophil Isolation Kit (Negative selection, Stem cell kit). On average, every 1 ml of blood yields 1.5–1×10⁶ cells (Additional file 1: Fig. S1A and S1B). Cells were maintained in 2% FCS in RPMI media (biological industries) supplemented with 1% penicillin/streptomycin/glutamine. Neutrophil baseline priming or activation status was evaluated by monitoring the rate of extracellular ROS release before or after phorbol myristate acetate (PMA) induction and the release of extracellular superoxide (Additional file 1: Fig. S1C). For this application, 1×10⁵ of highly pure neutrophils were isolated from whole blood samples from HC and HD patients. The cells were seeded in a 96-well black plate with PBS and 20mM of L-012 (Sigma-Aldrich) with or without stimulation with 100nM PMA in a total volume of 200μl at 37 °C. Rates of superoxide production were monitored by ELISA plate readers (SpectraMax Plus 384 Microplate Reader) at 510 nm for up to 30 min.

B16F10 melanoma cells stably express mCherry (herein B16F10), a kind gift from Prof. Ronit Satchi-Fainaro (TAU) were generated by infecting B16F10 cells with pQC-mCherry retrovirus. The cells were maintained in DMEM, supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 4 mM l-glutamine and 3 µg/ml puromycin at 37°C with 5% CO₂. RMS cells [25 (link)] were grown in RPMI media supplemented with 10% FCS, 1% sodium pyruvate and 5% penicillin-streptomycin (all from Biological industries, Beit-Haemek, Israel) at 37°C and 5% CO₂.
All cells were routinely tested for Mycoplasma using the EZ-PCR Mycoplasma Test Kit (Biological industries, Beit-Haemek, Israel).

The T24 cells were grown in McCoy’s 5A (Biological Industries) supplemented with 10% FBS (Capricorn Scientific). The cells were passaged twice per week, and the culture medium was changed with the same frequency. The LNCaP cells were maintained in RPMI media (Biological Industries) supplemented with 10% FBS (Capricorn Scientific) and 10% of its own conditioned medium (complete media). The cells were passaged once per week, and the medium was changed with the same frequency. The HaCaT cells (immortalized human keratinocytes) were cultured in Dulbecco’s modified Eagle medium (Biological Industries) supplemented with 10% FBS (Capricorn Scientific). The cells were passaged twice per week, and the medium was changed with the same frequency. The cell cultures were maintained under a humidified 5% CO₂ atmosphere at 37 °C.