Multi well culture plates

Non-axenic clonal cultures were established by isolation of Pseudo-nitzschia cells using drawn out glass pipettes (micropipettes) and a Nikon Eclipse TS100 inverted microscope (≤ 400x magnification) and transferred into 24 multi-well culture plates (Corning Inc. Durham, USA) containing 1 mL f/2 medium [39 ]. These well plates were kept at 16°C– 18°C under a photon flux of 60–100 μmol photon m^-2 s^-1 on a 12/12 hour dark/light cycle (white fluorescent tubes) and checked every alternate day. After 1 week, viable cultures were transferred to 70 mL gamma sterile polystyrene containers with polyethylene caps (Thermo Fisher Scientific, Australia, Pty.) and maintained in the same conditions. One milliliter of culture from each strain was transferred into fresh media every two weeks to establish healthy and exponentially growing monocultures over the duration of the study. On day 14 (late stationary phase) Pseudo-nitzschia cells were harvested for light (LM) and transmission electron microscopy (TEM) examination, DNA sequencing based on the large subunit (LSU) and internal transcribed spacer (ITS1-5.8S-ITS2) regions of the ribosomal DNA, and toxicity determination by liquid chromatography–mass spectrometry (LC-MS/MS) for the presence of DA.

Undifferentiated human monocytes (CRL-9855™) were purchased from ATCC® and sub-cultured in RPMI 1640 (Merck, Darmstadt, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% sodium pyruvate (all from Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO₂. Differentiated macrophages were obtained as previously described^{26 (link)}. Both the cell types were seeded at 0.5 × 10⁵ cells/well in multi-well culture plates (Corning, Falcon®, Glendale, Arizona, USA). To establish an inflamed environment, cells were stimulated with LPS 0.5 µg/mL (lipopolysaccharide from Escherichia coli, purchased from Merck, Darmstadt, Germany, stock solution 1 mg/mL in water). Untreated and LPS-stimulated samples were harvested immediately after treatment (T0 = time zero) and after 3 and 24 h and used for further procedures.