G-banding

Question 1

What is confluency of a culture?

Answer 1

A confluent culture is the optimum balance between health and number of the cells
After this the cells health will decline

Question 2

What are lymphoblastoid cells?

Answer 2

Derived from human B cells and infected with Epstein Barr virus
Grow as a suspension culture - do not adhere to the plastic

Question 3

What are HCT116 cells?

Answer 3

Male patient with malignant colonic carcinoma and is adherent - adhere to the flask
Heterogeneous = major population with 45, X and minor with 46, XY

Question 4

What is trypan blue?

Answer 4

Used to measure culture viability as dead cells have increased permeable membranes and trypan blue dye will enter the cell

Question 5

Why should over congruency be avoided for cell harvesting for G banding?

Answer 5

because they will not go into the cell cycle

Question 6

What is the centrosome?

Answer 6

A microtubule organising centre

Question 7

What are spindle fibres?

Answer 7

Microtubules from the centromere that attach to the kinetochore

Question 8

What phase do cells need tot be in for G-bandng?

Answer 8

Metaphase - condensed so regions can be resolved

Question 9

Why is it difficult to harvest cells in mitosis?

Answer 9

Only 2 hours of the cell cycle and asynchronous with the population

Question 10

What is colcemid?

Answer 10

At high concentrations it causes deploarisation of microtubules so cannot form mitotic spindles preventing cytokenesis

Question 11

What is the use of colcemid?

Answer 11

Used to arrest the cell population in metaphase and increase metaphases in cytogenetic harvest

Question 12

Why do cells need to be swelled in a harvest?

Answer 12

To increase the volume of the cell and spread out the chromosomes

Question 13

What is used to swell cells?

Answer 13

A hypotonic solution which has a lower concentration of salt than the cell allowing water to move in through osmosis
- KCl

Question 14

What happens when too much swelling?

Answer 14

Cells burst

Question 15

What is cell fixing?

Answer 15

Stabilising cellular morphology and inactivating biochemical activity

Question 16

What is used to fix cells for cytogenetics?

Answer 16

3 parts methanol and 1 part acetic acid
Methanol 0 dehydrates cell and permeabilise membranes
Acetic acids- denature proteins and counteracts methanol shrinkage

Question 17

How should fixative be added?

Answer 17

Slowly and constant while agitate culture

then change 2-3 times to further dehydrate without shrinkage

Question 18

Outline slide prep for G banding

Answer 18

Superfrost slides which can adhere cells should be washed in decon90 and stored at 4 degrees

Question 19

Why is slide dropping important?

Answer 19

facilitates the spreading of the genetic material and decreases background interference by cytoplasm components

Question 20

What is the ideal slide dropping height?

Answer 20

2-3cm

Question 21

What happens when the drop height is too high or too low?

Answer 21

Too low: poor spreading

Too high: cells burst

Question 22

Why does slide drying result in metaphase spreading?

Answer 22

The downward force of the liquid evaporating

Question 23

What happens when dry to quick or slow?

Answer 23

Too quick: Encapsulation (poor spreading)

Too slow: Burst due to prolonged stretching of membrane

Question 24

What happens when cells are encapsulated?

Answer 24

Poorly spread, chromosomal overlap and high background due to thicker cytoplasm layer absorbing light

Question 25

When does encapsulation occur?

Answer 25

Errors in harvesting or slide dropping, sweeping to fast fixation

Question 26

What is the mitotic index of a slide?

Answer 26

Number of metaphase compared to interphase

Question 27

What is slide ageing?

Answer 27

Exposed to sunlight for 48 hours to denature proteins, remove fixative and water

Question 28

What is leishmans stain?

Answer 28

A methylene blue stain which stains chromatin blue

Question 29

Outline G banding

Answer 29

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

Question 30

What effects quality of G banding?

Answer 30

Time, concentration and temperature of Trypsin addition