Cellstar

To analyze whether spraying influences the proliferation, an XTT assay (Roche Diagnostics) with n=5 was accomplished. Cells were sprayed in a 6-well plate (CellStar; Greiner Bio-One), and the suspension was then transferred into a 96-well plate (CellStar; Greiner Bio-One) for measuring. As a positive control, nonsprayed cells were pipetted into the wells; as a negative control, we used DMEM without fetal calf serum supplementation. On day 1, 3, and 6 after seeding, an XTT-reagent/coupling solution mixture (50:1) was added to each well; absorption was measured at 475 nm after 1 h of incubation at 37°C.

We established an embryonic A. ocellaris (EAO) cell line to analyse clock gene expression in living cells and facilitate further analysis, so not being dependent on sacrificing valuable adults. The EAO cell line was prepared using a method previously applied with zebrafish⁵⁶ . Eggs were scraped from one terracotta pot three days post fertilization and disrupted mechanically followed by 15 min incubation with Trypsin/EDTA (Gibco by Thermo Fisher Scientific, Paisley, United Kingdom). The cell suspension was transferred to a 6-well plate (Cellstar by Greiner Bio-One, Frickenhausen, Germany) and cultivated in Leibovitz’s L-15 medium supplemented with 15% fetal bovine serum (FBS), 100 U/ml penicillin/100 µg/ml streptomycin and 25 µg/ml gentamicin (all Gibco) at 27 °C. After one week, cell debris was removed and cells were washed with 1× PBS (Gibco) and were further incubated in culture medium. For passaging, a confluent cell monolayer was washed with 1× PBS, detached from the culture substrate by incubation with Trypsin/EDTA at 37 °C and then reseeded in culture medium in 25 cm² flasks (Cellstar by Greiner Bio-One).

Capability of MKs to form proPLT was studied as a functional parameter of iPSC-derived MKs, as previously described [14 (link),19 (link)]. Cells harvested at day 19 of differentiation, or cells directly after cryopreservation, were seeded on 12-well suspension culture plates (Greiner CELLSTAR^®; Greiner Bio-One, Kremsmünster, Austria) in APEL medium supplemented with 50 ng/mL of TPO and 50 ng/mL of SCF. After 24 h the images of MKs forming proPLTs were acquired using an Olympus IX81 microscope with a digital B/W camera (Olympus) and analyzed with the Xcellence Pro image software. ProPLTs were detected as elongated uniform tubular structures with periodic PLT-sized swellings [73 (link)].

The lysosomal function of cells was measured using a lysosomotropic tracking dye called LysoTracker® Red DND-99 (Life Technologies), which accumulates in lysosomes due to proton trapping (47 (link)). THP-1 macrophages or HMDMs (1 × 10⁶ cells per well in 12-well tissue culture plates) were incubated with prewarmed culture medium (2 ml per well) either alone or containing native LDL (100 μg protein/ml) or SMase-LDL (100 μg protein/ml) in the presence or absence of freshly dissolved cysteamine for 72 h at 37°C, with a change of medium every 24 h. After 72 h, the macrophages were washed three times with prewarmed PBS to remove any residual LDL or cysteamine. The adherent macrophages were scraped into culture medium using a plastic cell scraper, collected into 15 ml sterile polypropylene tubes, and centrifuged at 500 g for 5 min at room temperature to remove cell debris. The cells were resuspended into 200 μl RPMI-1640 medium [containing 10% (v/v) FCS], transferred into a clear 96-well round bottom microplate (Greiner CellStar®), and treated with LysoTracker Red (500 nM) in RPMI-1640 for 30 min at 37°C. Cells were washed twice with HBSS, resuspended in FACS buffer, and analyzed using a BD Biosciences C6 flow cytometer. The data analysis was done using FlowJo software by determining MFI for each histogram using untreated cells as a control.

In vitro biofilm formation capacity was determined for each isolate using a crystal violet assay. Briefly, all bacterial strains were incubated overnight on 5% sheep blood agar plates (bioMérieux, France) at 37°C and suspended in tryptic soy broth (TSB; Oxoid, ThermoFisher Scientific) to a concentration equivalent to a 0.5 McFarland standard. Ninety-six well polystyrene flat-bottomed microtiter plates (Cellstar®, Greiner bio-one®, Frickenhausen, Germany) were filled with 200 μL each and incubated at 37°C for 24 h. Planktonic cells were removed using phosphate-buffered saline (PBS), and biofilm was heat-fixed for 10 min at 60°C and subsequently fixed with 150 μl methanol (Merck, Darmstadt, Germany). Plates were dried at room temperature, stained for 20 min using 150 μl 1% crystal violet (Merck KGaA®, Darmstadt, Germany), and washed with tap water. For improved quantification, 150 μl 33% acetic acid (AnalaR Normapur, Prolabo®, VWR International®, USA) was placed in each well, and plates were incubated at 37°C and 50% humidity for 1 h. Plates were measured using a microplate reader (Sunrise, Tecan, Switzerland) at 595 nm with a reference measurement at 405 nm. The mean OD for each isolate was determined by measuring 14 replicates in two separate plates (7 replicates each).

Raji-cells were seeded in tissue culture flasks (Cellstar; Greiner Bio-One, Kremsmuenster, Austria) using RPMI-1640 medium supplemented with fetal bovine serum (FBS) to a final concentration of 10% (v/v). In order to do studies on cell binding 10 × 10⁶ cells were washed twice with fresh media, diluted with PBS to a final concentration of 1 × 10⁶ cells per mL and 500 µL of cell suspension was transferred to Eppendorf tubes. Hereafter, 50 µL of RTX-TCO or non-modified RTX as negative control (both 0.5 µM) were added and the cell suspension was maintained at 37 °C under gentle shaking. After 1 h the suspension was centrifuged (2 min, 11 × 10³ rcf), the supernatant was discarded, the cells were washed twice and finally resuspended with 450 µL PBS. Subsequently, 50 µL of the radioligand solution (22 nM in PBS) was added and the suspension was incubated for 30 min at 37 °C. After centrifugation and two washing steps with 600 µL PBS, the cells were resuspended in 500 µL PBS and transferred to polypropylene vials for gamma counter measurement followed by calculation from cell-associated activity in comparison to the standard (n = 3, six replicates).