Pipettes and Tissue Culture Flashcards
What are the 3 types of pipettes?
Pipettes
Pasteur pipettes
Micropipettes
Describe a pipette
A plastic of glass tube
Graduated to deliver any amount from 1-25mls
Suction of liquid via bub or pipette aid
Describe a Pasteur pipette
Small, tapered glass tube
Used with a bulb
Used to dispense liquid if volume not critical
Describe a micropipette
Use to dispense small volumes from 1-1000 microlitres
Used with disposable tips
How do you use a micropipette?
Depress the plunger to the first stop
Insert the tip into the liquid
Slowly release the plunger to pull liquid into the tip
Dispense by pushing the plunger to the second stop
How should a micropipette be held?
Hold vertically
What is molarity?
The measure of the concentration of a solute in solution
What is molarity measured in?
Moles
What are moles?
The molecular weight of a substance expressed in grams?
What is one mole of a substance?
Avogadro’s number of molecules of that substance
What does the weight/volume of a solution refer to?
The weight of a solute as a percentage of the volume
For aqueous solutions weight/weight is the same as weight/volume
T/F
T
When is volume/volume used?
To make a solution of two or more liquids
What does a “10X Buffer” mean?
You would need to dilute by 10 to get the 1x working concentration
(ie a final volume of 10ml needs 1ml of 10X buffer)
What is the formula for calculating dilution?
C1V1=C2V2
What is the goal of a lowry protein assay?
To use known concentrations to generate a standard curve
In order to calculate unknown protein concentrations
What is the Lowry protein assay?
Method involving copper bonding to measure protein concentrations through colorimetric techniques
Describe what happens in the lowry assay
Divalent copper ions form complexes with peptide bonds in the protein under alkaline condition
It is reduced then to a monovalent ion which reacts with Folin’s reagent along with radical groups of tyrosine, tryptophan and cysteine
This forms an unstable product reduced to molybdenum blue
Spectrophotometry used
How should a spectrophotometer be used?
Calibrate machine to appropriate wavelength
Set to zero with a control
Transfer liquid to cuvette (avoid touching the sides)
Place in spec and shut the lid
Read absorbance
What is the purpose of tissue culture?
To grow cells and passage them to allow propagation of the cell line
How can you continue growth of the cell line in tissue culture?
Provide cells with nutrients and a medium to grow in in vitro
This allows division by mitosis by the cells and the population can continue to grow
Until limited by nutrient depletion at which point they can be passaged
How many cell types do tissue culture dishes normally contain?
One
What is a “clone” population of cells?
A homogenous population of cells derived from a single parental cell
Give examples of applications for cell culture
Chemo-resistance testing
Genetic tests
Receptor type recordings
What is passaging?
Removal of medium and transfer of a proportion of the cells to a fresh growth medium
Why does passaging allow more growth?
Gives cells more space and fresh medium
In which phase do the cells not divide?
Lag phase
In which phase do the cells grow exponentially?
Log phase
What does the length of the lag phase rely on?
Growth phase of the cell line at the time of subculture and the seeding density
What is the role of PBS in tissue culture?
To buffer the solution
Reduce amount of Mg2+ and Ca2+ ions
What is trypsin?
A protease which interferes which adhesion proteins
What is the role of EDTA in tissue culture?
Reduces Ca2+ and Mg2+ as these normally enhance cell adhesion
Why do we want to detach the cells in tissue culture?
They cannot be passaged when stuck to bottom of dish
What was in the DMEM?
10% foetal bovine serum Glutamine 1% sodium pyruvate 1% pen/strep Phenyl red dye
What is the purpose of 10% foetal bovine serum in the DMEM?
Growth factors and attachment proteins
What is the purpose of the pen/strep in the DMEM?
To stop contamination
What is the purpose of the phenyl red in the DMEM?
Indicator
Why is PBS used to wash off the media?
Trypsin is inhibited by Mg, Ca and FBS in the media
Which component of the FBS affects the trypsin binding?
Alpha-1-anti-trypsin
What is trypsin?
A proteolytic enzyme serine protease
What is the role of trypsin?
Breaking bonds to detach cells from surface
What was the %CO2 of the incubator ?
5%
Diff per cell type - you get a guide with the cells
Why should you always passage your cells a couple of times before you do anything with them?
To establish they are healthy and growing ok
Establish no issues with freezing
Bulk up the cell number (not many are frozen)
