Softmax pro

Subconfluent BHK-21 cells in 96-well plates were infected with WT or mutant viruses at low MOI (0.2), resulting in conditions where spread through the culture and appearance of CPE and cell death occurred over approximately 2 days. The MOI for WT and mutant viruses was confirmed as equal by immunofluorescence. The appearance of CPE was recorded by capturing transmitted light images of each well every hour postinfection for 36 h using an imaging plate reader (SpectraMax MiniMax 300; Molecular Devices). The surface area covered by cells was automatically calculated for every image using image analysis software (SoftMax Pro; Molecular Devices). As cells died, the surface area covered became less, allowing quantitation of the rate of virus growth through the culture. Data were visualized using GraphPad prism.

Cells were fixed 4 h postinfection with 4% formaldehyde in PBS, permeabilized with 0.1% Triton X-100, and blocked with 0.5% bovine serum albumin (BSA) in PBS. The fixed monolayers were serially incubated with monoclonal antibody 2C2 (specific for FMDV nonstructural protein 3A) at 1:1,000 in block buffer, Alexa Fluor 488-conjugated anti-mouse secondary antibody (Invitrogen) at 1:200 dilution in blocking buffer, and the nuclear counterstain TO-PRO-3 (ThermoFisher Scientific). Immunofluorescence images were captured using an imaging plate reader (SpectraMax MiniMax 300; Molecular Devices). Quantitation of the total number of cells based on TO-PRO-3 fluorescence, number of infected cells based on Alexa Fluor 488 fluorescence, and intensity of signal for each cell was automated by image analysis software (SoftMax Pro; Molecular Devices). Data were visualized using Prism 7 (GraphPad).

Minimum essential medium (50 µL) supplemented with 25 mM HEPES, 1 g/L additional glucose, 1% MEM nonessential amino acids (100×), 0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions of 11 three-fold dilution steps covering a range from 100 to 0.002 μg/mL were prepared. Then, 4 × 10³ bloodstream forms of T. b. rhodesiense STIB 900 in 50 µL was added to each well and the plate incubated at 37 °C under a 5% CO₂ atmosphere for 70 h. Ten microliters of resazurin solution (resazurin, 12.5 mg in 100 mL double-distilled water) was then added to each well and incubation continued for an additional 2–4 h [24 (link)]. The plates were then read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analyzed with the graphic program Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA), which calculated IC₅₀ values by linear regression [23 (link)] and 4-parameter logistic regression from the sigmoidal dose inhibition curves. Melarsoprol (Arsobal Sanofi-Aventis, received from WHO) was used as control.

Human chondrocytes C28/I2 were used as model cells to carry out the in vitro cellular studies. Cells were cultured in 75 cm flasks in Dulbecco’s Modified Eagles’ Medium (DMEM) supplemented with fetal bovine serum (FBS) and 1% antibiotics (penicillin–streptomycin) at 37 °C and an atmosphere of 95% air and 5% CO₂.
To determinate the cytocompatibility of CS-PF and CS-PF-TPP hydrogels against C28/I2 cells, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium (MTS) assay was performed. A piece of dried hydrogel sample was placed in a 96-well plate, and cell suspensions (1 × 10⁴ cells/well) in Dulbecco’s modified Eagles medium (DMEM) were seeded on the hydrogel. To establish the cytotoxicity controls, cells treated with 50% DMSO and non-treated were used as positive and negative controls respectively. The MTS assay was performed at 1, 2, 3, and 7 days. At each time point, 20 µL of MTS was added and incubated in darkness for 3 h. After that, 100 µL of the supernatant was extracted to a new 96-well plate. The absorbance of each well was measured by a micro-plate reader (VersaMax equipped with Softmax Pro, Molecular Devices, San José, CA, USA) at 490 nm. Cell viability is expressed as the percentage of cells alive in relation to negative control (cells incubated only with DMEM culture medium were used as a negative control (100%)).