LEAD

Antiretroviral efficacy

Goal: Evaluation of compound efficacy against HIV-1 infection of cell cultures, in vitro.

Methods:
      – 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.

Antibiotic efficacy

Goal: Evaluation of antibiotic efficacy against planktonic bacteria and pre-formed biofilms, in vitro.

Methods:
      – 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

Vision Sciences

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

Tissue culture:
      – Organotypic retinal cultures from mouse, rat and human

Cell lines:
      – Endothelial cells (HUVEC)
      – Retinal Müller cells (MIO-M1)
      – Microglial cells (BV-2)

Injury models:
      – 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