Notes 20100429 Catherine Group Meeting Proteomics Chinese Hamster Ovary Apoptosis
From SnOwy - Ed's Wiki Notebook
Catherine is practicing her Master's thesis defense talk.
Contents |
Comparative Proteomics, Prolonged Cultivation
- speaker: Catherine Wei
- CHO cultures
- differentially expressed proteins
- CHO cultivation
- Chinese Hamster Ovary Cells (CHO cells)
- cell line: monoclonal anti-body used-- IgG-9β8
- protein manufacturing with CHO
Apoptosis
- accounts for majority of cell death in bioreactors
- true in pharmaceutical productions
- observed in particular for CHO cell lines
- apoptotic bodies are removed by macrophages or just lyse in vitro
- intrinsic pathway: intracellular stress stimuli
- extrinsic pathway: some signal
- both pathways lead to activation of caspase 3
Objectives
- proteomic analysis of CHO cells at different phases of apoptosis
- non-induced apoptosis examined -- first study to characterize intrinsic apoptosis proteome in mammal culture
- flow cytometry
- fluorescent inhibitor for caspase (FLICA)
- for activated caspases 3
- propidium iodide (PI) -- membrane permeability (= viable) stain
- four groups
- live non-apoptotic
- live apoptotic: activated caspases
- apoptotic dead: activated caspases, propidium iodide
- degraded dead: propidium iodide
CHO cell growth
- three parts to the curve: an exponential phase, a stationary phase, a death phase
Protein samples, two week analysis
- collected during the two week analysis
- throughout the time course, the time points between phases were identified
- suspect that protein signature changes
- DIGE used at the phase boundaries
- cytoskeletal protein expression also characterized
