Cell Culture Flashcards
Cell culture history: Early 1900s
Harrison’s tissue culture methodology published
Cell culture history: 1950s
Spurred by virology (polio), heavy investment at establishing modern cell culture techniques
Cell culture history: 1970s
Recombinant DNA technology and hybridoma cells broaden applications
What kind of cell lines are there?
- Primary and continuous
- Adherent and suspension
What are some cell culture concepts?
- Mimicking cellular environment
- Extracellular fluid
- Nutrients
- Confluency
What are some applications of cell culture?
- Investigate cell physiology and/or biological function of a single gene product
- Perform hypothesis-driven systems biology research
- Synthesize “biologicals” (e.g., vaccines, recombinant proteins)
- Monoclonal antibodies for basic science or therapeutic use
What is the significance of mammalian cell culture for the study of PTEN?
- Visualize PTEN localization in human cells
- Characterization of PTEN on cellular processes
- Test mutations for affect on PTEN function
- Capture novel interacting protein prior to MS using affinity chromatography
What are Hek 293 cells?
Human embryonic kidney cells (designation 293)
What are cell culture techniques?
- Trypsinize pre-initiated stock cell plate
- Quantify cell numbers
- Plate specific amounts into two plates that will be used for transfection step
- Perform 1/10 split to keep a stock plate going
What are overexpression techniques?
- Transfection of FLAG-PTEN into Hek 293
- Cell lysis
What do detergents do?
React and destroy the cell membrane so intracellular components are released
What are the steps for detergent cell lysis?
1) Remove media
2) Wash with cold PBS
3) Lyse with detergent containing buffer - pipette a few times to dislodge cells from plate
- Centrifuge and keep supernatant
- Label appropriately and place in freezer
