Goal: Evaluation of compound efficacy against HIV-1 infection of cell cultures, in vitro.
– Infection-triggered phenotypic assay in TZM-bl cell line.
– Luciferase bioluminescence quantification.
– Time-of-compound-addition assay.
Deliverables: HIV-1 infection inhibition dose-response curves and respective endpoints. Stage of antiretroviral action.
Goal: Evaluation of antibiotic efficacy against planktonic bacteria and pre-formed biofilms, in vitro.
– Colorimetric viability assay (resasurin);
– Crystal violet assays (biomass quantification);
– Growth inhibition (MIC) assay;
– Bactericidal (MBC) assay;
– Colony counts assay.
Deliverables: Minimum inhibitory concentrations. Minimum bactericidal concentrations. Biofilm integrity and bacteria viability dose-response curves.
IN VITRO EFFICACY AND TOXICITY TESTS USING PRIMARY CELL CULTURES, CELL LINES, TISSUE CULTURES AND INJURY MODELS
Primary retinal cell cultures:
– Retinal mixed cultures
– Retinal ganglion cell cultures
– Retinal Müller cell cultures
– Retinal microglia cultures
– Bovine retinal endothelial cell cultures
– Porcine retinal pigment epithelial cell cultures
– Organotypic retinal cultures from mouse, rat and human
– Endothelial cells (HUVEC)
– Retinal Müller cells (MIO-M1)
– Microglial cells (BV-2)
– Elevated glucose concentration (model of diabetic retinopathy)
– Elevated Hydrostatic Pressure (EHP) (model of glaucoma)
– Excitotoxicity with N-methyl-d-aspartate (NMDA) or glutamate (model of retinal neurodegeneration)
– Exposure to lipopolysaccharide (LPS) (inflammatory model)
– Endothelial cells exposed to cytokines (breakdown of blood retinal barrier model)
Assays and Techniques:
– Cell viability assessment
– Reactive oxygen species (ROS)
– Griess reaction assay (NO)
– Cell proliferation assay
– Scratch wound assay and boyden chamber Migration assay (cell migration)
– Phagocytosis assay
– Cell death assays
– Angiogenesis assays
– Permeability assays
– Leukocyte adhesion to the endothelium assay
– Single cell intracellular calcium imaging