6 well culture plates

Two antiseptics were tested using a time-kill-assay investigating their antimicrobial efficacy on biofilm-growing bacteria: Octenisept (0.1% octenidine-dihydrochloride/2% phenoxyethanol; Schülke & Mayr GmbH, Norderstedt, Germany) and Lavasorb (0.04% polyhexanide; Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany). For this analysis, hpBIOMs with MRSA or P. aeruginosa were prepared in 6-well culture plates (Sarstedt AG & Co., Nürnbrecht, Germany). After either 12 h or 24 h of undisturbed biofilm maturation, biofilm models were treated with 600 µl of either antiseptic solution and incubated on a rotation shaker (50 rpm) at RT. Every 24 h, the same dosage of antiseptic solution was re-administered (mimicking a daily antiseptic treatment in clinical routine). Controls were treated with PBS only. After 6, 24, 48 and 72 h of exposure, antiseptic activity was terminated using a neutralization buffer containing 3 g/l sodium thiosulfate, 30 g/l polysorbate 80 (Tween 80) and 3 g/l lecithin (all Carl Roth GmbH u. Co. KG, Karlsruhe, Germany). Previously performed validations confirmed no antimicrobial or disintegrative effect on the biofilm model as well as sufficient neutralizing capability of the neutralization buffer.

Plasma preserves and buffy coats from anonymous donors were obtained from the DRK-Blutspendedienst West (Hagen, Germany). First, the buffy coat was transferred to a 50 ml reaction tube (Sarstedt AG & Co., Nürnbrecht, Germany) and centrifuged at 3000 rpm for 30 min at room temperature (RT) to separate remaining erythrocytes. The resulting supernatant of blood plasma and layer of immunocompetent cells was collected and added to the plasma preserve of the same donor in a sterile glass bottle. The resulting mixture of plasma and buffy coat was gently mixed and continuously shaken at 100 rpm at 22 °C. Subsequently, a bacterial solution of the individual test pathogen was prepared by colony-picking and the solution adjusted to a 0.1 McFarland standard. For final preparation a corresponding amount of prepared bacterial solution was added to the plasma/buffy coat mixture to result in initial pathogen concentration of 2*10⁶ cfu per 3 ml plasma solution, creating a master mixture of plasma, buffy coat and pathogen. To induce fibrin polymerization (for disc-formation), 18.3 µl CaCl₂ (500 mM) per ml plasma was added to the master mixture. Finally, to prepare individual hpBIOMs, 3 ml master mixture per well were immediately transferred into 6-well culture plates (Sarstedt AG & Co., Nürnbrecht, Germany). The plates were incubated for at least 12 h on a rotation shaker at 50 rpm at 37 °C.

hAoSMCs (catnr: C-007-5C, lot: 2164581, Life Technologies, Carlsbad, California, USA and Cell Applications Inc. San Diego, California, USA) were cultured in Medium 231 (Life Technologies) supplemented with smooth muscle growth supplement (complete SMC medium) (Life Technologies) and 10 U/ml penicillin and streptomycin (PEST) (Life Technologies) at 37 °C and 5% CO₂. The medium was changed every 48 h and the cells up to passage 6 were sub-cultured or used for experiments upon 80–90% confluency by detachment with trypsin (Life Technologies). hAoSMCs were seeded at a density of 1.25 × 10⁵ cells/well in 6-well culture plates (Sarstedt Inc., Nümbrecht, Germany) overnight and subjected to different treatments for various timepoints on the following day. Cells and supernatants were stored at − 80 °C until further analyses.

HEK293 cell line was grown in DMEM (glucose 1 g/L, Merck, Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% (v/v) inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Carlsbad, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mM glutamine (Thermo Fisher Scientific, Invitrogen, Carlsbad, CA, USA). HEK293 cells, 5 × 10⁵ cell/well in 6-well culture plates (Sarstedt, Hildesheim, Germany), were transfected with 2 µg cDNA of the plasmid carrying the different PCSK9 variants using the calcium phosphate transfection method. Similar transfection efficiency was confirmed by transfecting in parallel a plasmid encoding a green fluorescent protein.

The Nicotiana tabacum cv. Bright Yellow 2 (tobacco BY-2) cell suspension was made available by Toshiyuki Nagata (Tokyo University, Tokyo, Japan) and cultivated at 26 °C, on a rotary shaker set at 140 rpm in the dark, in modified Murashige and Skoog (MS) medium as reported [42 (link)]. For treatments, 7-day old cells were diluted five-fold into fresh MS medium and distributed (3 mL) in 6-well culture plates (Sarstedt, Nümbrecht Germany) containing 25 or 50 µM of VH, Mad, Emo, Qui, Fra or Lap. SImaging acquisitions were carried out either after 5 min incubation or after 18 h.