protocol 6-trypan blue exclusion assay and cell count

the development of automatic cell encounters

has made hemocytometers obsolete in many places

hemocytometers offer the user being able to

identify cells and distinguish between living and dead cells

what’s this organic molecule

trypan blue

trypan blue is also what

a chromophore

chromophore

only bind to damaged membranes

chromophore is internalized by what

dead or dying cells, but excluded from viable cells

how can trypan blue be used

to distinguish between living and dead cells by only nonviable cells being lysed and taking this into the cell

how to determine the percent of living and dead cells in a cell culture

trypan blue, placing sample of cells on hemocytometer slide, and then using a microscope to count cells

how is hemocytometer an aid

it contains a grid to help with cell counting

total number of viable cell/ml formula

average cell count per square x dilution factor x conversion factor

include what cells in counting

bottom and left hand perimeters

what cells not to include in cell counting

top and right hand perimeters

trypsin

enzyme that partially degrades the extracellular proteins responsible for adherence of cells on disk/flask surface

what is trypsin inactivated by

serum

prior to adding the trypsin what must happen

to remove all traces of serum in the media

what else inactives trypsin

addition of serum-containg media after the minute incubation

BSS

an osmotically balanced aqueous salt solution

BSS pH

7.4

PBS (phosphate buffered saline)

example of common BSS used in cell culture

percent viability formula

(total viable cells/total viable+non viable) x 100