48 well plate

For assessing the anti-Aspergillus activity, MSCs were incubated in 48-well-plates (Nunc) overnight. After washing, cells were co-incubated with 1000 conidia in 1 mL medium at different effector:target ratios. After 4 hours, 100 μL were spread on a Sabouraud glucose agar plate (Becton Dickinson, Heidelberg Germany) and incubated overnight. The anti-conidial effect was assessed by comparing the average number of colony-forming units (CFUs) with and without co-incubated MSCs, respectively. Cytochalasin D (1 μmol/L) and colchicine (2 μmol/L) (Sigma-Aldrich) were used as phagocytosis inhibitors as described previously [20 (link)].

Peripheral blood mononuclear cells (PBMCs) were prepared from heparinized blood by standard Ficoll–Paque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden). Cells were cultured in RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA) containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (Gibco BRL) that had been inactivated by heating to 55 °C for 30 min. Suspensions of both cell types were dispensed into 48-well plates (Nunc, Rosklide, Denmark). PBMCs were incubated with plate bound anti-CD3 (0.5 µg/mL) or lipopolysaccharide 100 ng/mL for 3 days.

Adult worm drug testing was performed as previously reported [29] (link) with some modifications as described. Worms of a Puerto Rican strain of S. mansoni were obtained by portal perfusion of CD1 mice (Charles River, UK) 6 weeks post-infection. Three pairs of worms were added to the wells of 48-well plates (Nunc, UK) in 1 ml complete DMEM medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (cDMEM). Compounds were tested at 15 µM containing 0.15% DMSO in single wells. Negative controls contained worms cultured in cDMEM alone and in cDMEM with 0.15% DMSO. Positive control wells contained worms cultured in praziquantel (Sigma-Aldrich, UK) at 10 µM. Cultures were incubated at 37°C and 5% CO₂. Effects were assessed on day 5 of culture using an inverted microscope (Leitz Diavert Wetzlar, Germany). Any compounds producing complete immotility or ≥70% worm motility inhibition plus severe morphological damage were considered hits in the primary screen [29] (link). Active compounds were then tested for IC₅₀ value determination at a concentration range from 0.55–15 µM in single wells.

Liver cells (stock: 10⁷/ml) were diluted till 2×10⁶ cells/ml in RPMI-1640 medium/10% FCS/non-essential amino acids/glutamate/penicillin/streptomycin. Next, 500 µl cell suspension/well were cultured (36 hours, 37°C, 5% CO₂) in 48 well plates (Nunc) and the supernatant tested for TNF levels.

The T84 cell assay was performed essentially as described previously [26 (link)]. Briefly, T84 cells (ATCC, Rockville, MD, USA) were seeded and grown to confluence on 48-well plates (Nunc, Roskilde, Denmark) in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) (Gibco life technologies, Paisley, UK), supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 0.2% gentamicin (LONZA, Walkersville, MD, USA). The cells were washed 3 times with 250 µL DMEM-F12 and pre-incubated with 40 µL DMEM-F12 containing 1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) for 10 min at 37 °C. A volume of 40 µL 1 µM native or mutant STh peptide was added to each well in duplicate (final peptide concentration 0.5 µM) and incubated for 30 min at 37 °C. Following incubation, the reaction medium was aspirated, and the cells were lysed with 0.1 M HCl at 20 °C for 20 min. Subsequently, the lysates were centrifuged at 16,000× g for 10 min, and the supernatants were collected to estimate cGMP levels using a cGMP enzyme immunoassay kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA). The analysis was conducted according to the manufacturer’s instructions.

The cell viability and proliferation were assessed using the CCK-8 assay kit (Dojindo, Rockville, MD, USA) at 0, 1, 2, and 3 days. hMSCs were seeded into 48-well plates (Nunc, Roskilde, Denmark) at a density of 1 × 10⁴ cells/mL. Each plate was pre-incubated in a humidified incubator with 5% CO₂ at 37 °C, and 20 μL of the CCK-8 solution was added to each well and then incubated for 2 h. 100 μL/well aliquots were transferred to a 96-well plate (Nunc) and absorbance was measured at 450 nm using an Opsys MR micro-plate reader (DYNEX Technologies Inc., Denkendorf, Germany). The cell viability was determined in 24 h absorbance data. The cell proliferation rate was assessed in optical density (OD) units.