Cellular pharmacology

Accessible equipment (SERCOBIO)

- Flow cytometry and cell sorting facility

• Contact: Anabelle Sequeira-Le Grand, MD
  Place: IFR100 Université de Bourgogne - Faculté de Médecine - 7 Bld Jeanne d'Arc - 21000 Dijon
• Elite-Epics ESP (Beckman-Coulter) for cell analysis and cell sorting: 5 photomultipliers and 3 lasers (488 nm/15 mW, 633 nm/10 mW and Laser Argon ion U.V. 350 nm/25 at 150 mW)
• Flow Cytometer LSRII (Becton Dickinson) for cell analysis: 10 photomultipliers and 3 lasers (5 PMT laser 488 nm/20 mW, 3 PMT laser 633 nm/17 mW and 2 PMT laser U.V. 325 nm)
• 1 PC for data acquisition
• 1 PC for data analysis
• Cell cycle analysis software (Multi Cycle)
• Multi TIME software for ion flow analysis (Ca2+, K+)

- Center of Microscopy applied to Biology (CMAB)

• Contact: C. Humbert (Director) and Dr. G. Lizard
  Place: ENSBANA Campus Universitaire Esplanade Erasme 21000 Dijon
• HITACHI 600 transmission electron microscope
• JEOL JSM 35 scanning electron microscope with thermo-regulated sample holder
• LEICA TCS 4D confocal laser scanning electron microscope
• LEICA cryo-ultramicrotomy equipment
• Critical point drying equipment

Resource centre for biological materials

• Tissues (Hôpital du Bocage, Centre de Lutte contre le Cancer Georges-François Leclerc...)
• Whole blood and blood fractions from healthy donors (EFS Bourgogne Franche-Comté)
• Cells (ATCC, DSMZ, Ludwig Institute Brussels...)

Experiments performed by the laboratory.

• Primary and tumor cell culture
• Culture of adherent cells, suspension cells, spheroids, co-culture, semi-solid medium
• Establishment of cell lines
• Cloning
• Production of cells and cellular extracts
• Trypan blue exclusion
• Crystal violet, methylene blue and neutral red staining
• Mycoplasma testing
• Alamar blue test
• MTT test
• SRB test
• ⁵¹Cr or calcein release
• Intracellular calcium mobilization assay
• BrdU incorporation
• Double agar layer clonogenic assay
• Annexin V labeling
• Propidium Iodide labeling
• TUNEL labeling (DNA fragmentation)
• Hoechst staining (DNA condensation)
• Marker analysis using FACS, fluorescent microscopy, ELISA (assay development required)
• Transient and stable transfection of cells, selection and characterization of stable clones
• Development and isolation of resistant cell lines
• Boyden chamber assay for chemotactic response (assay development required)
• Cell invasion test
• Scraping test
• Agar drop migration test
• Development, transfer and validation of cellular tests

Aim of these experiments in drug development

Evaluation of anti-proliferative activity, cytotoxic activity, drug combinations, screening (MTT, SRB, BrdU, ⁵¹Cr or calcein release, clonogenic assays)

Approach of the mechanism of action

• Dispatching of cells in cell cycle phases (BrdU, PI incorporation)
• Induction of cell death: detection of apoptosis, necrosis (Annexin V, TUNEL, Hoechst, Sub G1, DIOC6)
• Cellular migration (chemotactism, scraping, cell injury)
• Differentiation (expression of markers, morphology)

Model characterization

• Expression of cellular markers (Western Blot, ELISA, FACS)
• Subcellular localization (Microscopy)

Model development

• Transfections
• Resistant cell lines
• Target-based assays
• Intracellular Ca²⁺ release
• Production of ATP
• Production of ROS
• Enzymatic activity