Viability Assays Flashcards
What is the solution we grow cells in called?
Growth medium
What does growth medium contain?
amino acids, vitamins, salts & sugars
How do we supplement protein into cell culture?
- using fetal calf serum
Describe Fetal Calf Serum
- fraction purified from blood of bovine calves
- non definded mixture of proteins, growth factors & cytokines
What flasks are typically used in cell culture?
T25s & T75s
What is the base area of a T25 flask?
25cm ^2
What is the base area of a T75 flask?
75cm^2
What does anchorage dependant mean?
- many human cells are anchorage dependant, meaning they need to stick to the base of the flask to grow
How do cells adhere to the flask?
- cells secrete ECM
How do we remove adhered cells from flask walls?
- need to destroy the ECM without damaging the cells
- this is carried out using Trypsin
What is the biggest risk to tissue cultures?
Microbial infection
What is included to help alleviate infection in growth culture?
penicillin & streptomyocin
Define Viability
- capable of living
- capable of growing/developing
Describe Trypan Blue
- diazo dye -> its a dye exclusion method
- routine test to determine percentage viability of a populations of cells
- viable = alive
- percentage viability tells us what proportion of cells are dead & alive
How does Trypan Blue enable us to calculate viability?
- cells which have taken up the dye are blue & we know are dead
- not taken up dye = colourless & are alive
Describe the principle of Dye Exclusion
- principle that intact membrane of a living cell will exclude a given substance (dye)
- when cells dye the membrane becomes compromised & substances that were once excluded can now traverse the membrane
Describe a Haemocytometer
- modified microscope slide with a counting grid etched into it
- liquid dispensed onto gird using a pipette & seal with coverslip
What is the equation for percentage viability ?
(live cells/ total cell number ) x 100
