Subcloning and Enumeration Flashcards
NIH3T3 species, tissue origin, growth properties, morphology, karyotype
Mus musculus NIH/Swiss embryo adherent fibroblastic- stellate diploid, 40- no longer diploid
HeLa species, tissue origin, growth properties, morphology, karyotype
Homo sapiens cervical carcinoma from 31 yr old female adherent, suspension epithelial aneuploid 81-83, XX
CMF-PBS purpose
maintains proper pH and osmotic balance
washes away protease inhibitors contained in serum
loosens cell to cell contact to permit trypsin access to the lateral and basal cell surfaces
Trypsin-EDTA purpose
enzyme that cleaves between adjacent arginine and lysine residues in protein (ECM)
EDTA chelates divalent cations
neutralized by addition of sera
Viability stains
exclusion dyes
the loss of cell membrane integrity allows the dye to enter the cell
Viability stains examples
trypan blue (stains nonviable cells blue) eosin y (stains nonviable cells red) propidium iodide (stains DNA and RNA fluorescent red)
Errors common with hemocytometer
improper filling of chambers (over- or under- filling)
too many cells (between 20 and 50 cells/square)
non-homogenous suspension
variable sampling of suspension
inconsistent pattern of counting
statistical error
FLOW cytometry
a bean of laser light of a single frequency is directed onto a hydrodynamically focused street of fluid, detectors are aimed at the point where the stream passed through the light beam; 1 in line (forward scatter) and many perpendicular (side scatter)
Forward scatter
the magnitude of the forward scatter is roughly proportional to the size of the cell
Side scatter
the granularity of the cell and internal complexity of cell influence the degree of side scatter
Propidium iodide
intercalates into nucleic acids and can penetrate the cell membrane of dead or dying cells
