24 well plate

Explants were cultured with 1 mL medium at 37 °C and 5% CO₂ in 24 well plates (IWAKI, Willich, Germany). D-MEM (4.5 g/L glucose; Life technologies, Vienna, Austria) supplemented with 100 U/mL nystatin (Life technologies) and 0.1 mg/L gentamicin (Life technologies) was used as the culture medium. Explants were cultured with different FB₁ concentrations (0–10 µg/mL) for 24 h (quadruplicate per horse) or 48 h (triplicate per horse). As positive control, explants were incubated with 10 µg/mL lipopolysaccharides from Escherichia coli O55:B5 (Sigma, Vienna, Austria) [34 (link)] for 24 h (quadruplicate per horse). Thereafter, all explants were examined via microscope before proceeding with testing. Explants showing bacterial or fungal contamination were excluded from the results.
Explants were tested for their viability with the water soluble tetrazolium (WST-1) as described by Reisinger et al. [34 (link)]. A comparison between control explants (0 µg/mL FB₁) for 24 (n = 12) and 48 h (n = 9) was performed. The force required for explant separation was measured by a calibrated force transducer as described by Reisinger et al. [34 (link)].

HEK293 cells were seeded on 24-well plates (Iwaki) and transfected with designated plasmids. After 24 hours post-transfection, cells were lysed in 1× Passive Lysis Buffer (PLB; Promega). Each well was added with 100μl 1×PLB and lysed for 30 min at room temperature. 30μl of cell lysate was then transferred to the 96-well plate. The Firefly and Renilla luciferase activities were measured by the Microplate Luminometer LB 96V (EG&G Berthold, MicroLumat Plus) by Dual-luciferase Reporter Assay System (Promega).

HEK293, HEK293T, HEK293M81, HEK293p2089 and HEK293p2089-C61A cells were seeded onto 60mm dishes, 6-well or 24-well plates (Iwaki) at a concentration of 1 × 10⁵ per ml and transfected with GeneJuice (Novagen) 24 hours later in a ratio of 1μg DNA to 3μl GeneJuice. ISD90 (4 μg/ml) was transfected into HeLa cells in a 1 μg DNA to 3 μl Lipofectamine 3000/P3000 ratio according to manufacturer’s instruction. ISD90 primers were 5′- TACAGATCTA CTAGTGATCT ATGACTGATC TGTACATGAT CTACATACAG ATCTACTAGT GATCTATGAC TGATCTGTAC ATGATCTACA (forward) and 5′- TGTAGATCAT GTACAGATCA GTCATAGATC ACTAGTAGAT CTGTATGTAG ATCATGTACA GATCAGTCAT AGATCACTAG TAGATCTGTA (reverse).

HeLa cells (8 × 10⁴ cells/well) were seeded onto 24-well plates (Iwaki) and cultured at 37 °C with 5% CO₂ for 24 h. Cells were washed twice with PBS(–). To evaluate the cellular internalization pathway of NLS-(− 30)GFP-LNP, HeLa cells were pre-treated with endocytosis inhibitors, 30 μM of Pitstop2 (a clathrin-mediated endocytosis inhibitor), 80 μM of 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a macropinocytosis inhibitor), or 500 nM of wortmannin (a macropinocytosis-related PI3K inhibitor) in α-MEM(–) for 0.5 h at 37 °C. Next, NLS-(− 30)GFP-LNPs (equivalent to 2.5 μM NLS-(− 30)GFP) in α-MEM( +) were added to HeLa cells and incubated with the endocytosis inhibitors for 1 h at 37 °C. The cells were then washed twice with PBS(–) containing 0.5 mg/mL heparin and incubated with 0.01% trypsin for 10 min at 37 °C. Suspended cells were collected in 1.5 mL tubes, washed twice with PBS(–), and subjected to flow cytometry analysis using an Attune NxT flow cytometer (Thermo Fisher Scientific). The analysis was performed on 10,000 gated events per sample.