G-banding Flashcards
What is confluency of a culture?
A confluent culture is the optimum balance between health and number of the cells
After this the cells health will decline
What are lymphoblastoid cells?
Derived from human B cells and infected with Epstein Barr virus
Grow as a suspension culture - do not adhere to the plastic
What are HCT116 cells?
Male patient with malignant colonic carcinoma and is adherent - adhere to the flask
Heterogeneous = major population with 45, X and minor with 46, XY
What is trypan blue?
Used to measure culture viability as dead cells have increased permeable membranes and trypan blue dye will enter the cell
Why should over congruency be avoided for cell harvesting for G banding?
because they will not go into the cell cycle
What is the centrosome?
A microtubule organising centre
What are spindle fibres?
Microtubules from the centromere that attach to the kinetochore
What phase do cells need tot be in for G-bandng?
Metaphase - condensed so regions can be resolved
Why is it difficult to harvest cells in mitosis?
Only 2 hours of the cell cycle and asynchronous with the population
What is colcemid?
At high concentrations it causes deploarisation of microtubules so cannot form mitotic spindles preventing cytokenesis
What is the use of colcemid?
Used to arrest the cell population in metaphase and increase metaphases in cytogenetic harvest
Why do cells need to be swelled in a harvest?
To increase the volume of the cell and spread out the chromosomes
What is used to swell cells?
A hypotonic solution which has a lower concentration of salt than the cell allowing water to move in through osmosis
- KCl
What happens when too much swelling?
Cells burst
What is cell fixing?
Stabilising cellular morphology and inactivating biochemical activity
What is used to fix cells for cytogenetics?
3 parts methanol and 1 part acetic acid
Methanol 0 dehydrates cell and permeabilise membranes
Acetic acids- denature proteins and counteracts methanol shrinkage
How should fixative be added?
Slowly and constant while agitate culture
then change 2-3 times to further dehydrate without shrinkage
Outline slide prep for G banding
Superfrost slides which can adhere cells should be washed in decon90 and stored at 4 degrees
Why is slide dropping important?
facilitates the spreading of the genetic material and decreases background interference by cytoplasm components
What is the ideal slide dropping height?
2-3cm
What happens when the drop height is too high or too low?
Too low: poor spreading
Too high: cells burst
Why does slide drying result in metaphase spreading?
The downward force of the liquid evaporating
What happens when dry to quick or slow?
Too quick: Encapsulation (poor spreading)
Too slow: Burst due to prolonged stretching of membrane
What happens when cells are encapsulated?
Poorly spread, chromosomal overlap and high background due to thicker cytoplasm layer absorbing light
When does encapsulation occur?
Errors in harvesting or slide dropping, sweeping to fast fixation
What is the mitotic index of a slide?
Number of metaphase compared to interphase
What is slide ageing?
Exposed to sunlight for 48 hours to denature proteins, remove fixative and water
What is leishmans stain?
A methylene blue stain which stains chromatin blue
Outline G banding
Aged slides are exposed to trypsin which degrades histone proteins
Condensed heterochromatin hide some of the histones preventing degradation
Then treated with leishmania stain which stains these heterochromatic regions darker as they are more intact than digested euchromatic regions
What effects quality of G banding?
Time, concentration and temperature of Trypsin addition
