MICRO LAB FINAL REVIEW

- Cell line and proper conditions

- Your compound
- Overall purpose
- Controls
o H202
 Kills cells (positive control for EC50)
o Ceramide
 Blocks cell migration (positive for wound closure)
o Vinblastine
 Blocks tubulin polymerization (positive for cell cycle, inhibits spindle
assembly in mitosis metaphase)
- Assay 1
o Protein titration curve
o Learn about micropipettes (P20 has decimal)
o Needed to find the unknown concentration w absorbances and known
concentrations
- Assay 2 (crystal violet)
o Based on color (qualitative)
o Purple=living cells bc it binds to the DNA
o Dead cells clear
- Assay 3
o EC50- concentration at which there will be 50% living and 50% dead
o Quantitative
o If membrane is integral then died by Hoescht bc it can go through the membrane
o PI is NOT permeable meaning that it can only stain the dead cells
- Assay 4: Cell migration assay (wound healing)
o Metastasis
o Primary vs secondary
 Primary: specific location
 Secondary: migrated elsewhere (metastasis)
o Test if compound would stop cell migration
o This assay used 5% FBS instead of 10%
 Used to slow the growth in order to observe whether the cells moved or
not, but still needed to give the nutrients necessary for it to grow
- Assay 5: Cell cycle
o 4 different checkpoints in cell cycle, and each for different phase
- Interphase:
o G1: growth phase, restriction point, ensured that cells have enough growth
factors to continue the cell cycle. If nutrients not available, they would go back
to receive proper nutrients or they die.
o S phase: DNA replication, DNA damage point. Ensure that DNA replicated
correctly (free of errors)
 o G2 phase: second growth phase, DNA damage checkpoint. Ensure that DNA is
okay and cells are ready for mitosis.
- Mitosis
o Prophase
 Condensation of chromatin to chromosome
o Metaphase
 Spindle check point
 Chromosomes are lined up in the middle of the cell
o Anaphase
 Spindle fibers bring back the chromosomes
o Telaphase
 The cells are separated
- Cytokinesis:
o Not part of mitosis, they get completely separated
- Used to see where the cells would stop in the cell cycle, at what checkpoint would they
stop?
- Asynchronous population
o Not all cells are in the same part of the cell cycle
- Diploid
o 2n (2chromosomes)
- Tetraploids
o 4n (more chromosomes)
Vehicle was DMSO ALWAYS

MATH
- C1V1=C2V2
o Initial concentration, initial volume, final concentration and final volume
o