Cytogenetic harvesting

Question 1

What are the steps of harvesting cells for cytogenetic analysis?

Answer 1

• prep and culturing of cell samples
• disruption of microtubules within cells
• dwelling of cells using a hypotonic solution
• fixing of cells using an alcohol/acid solution
• storage of fixed samples at -20°C

Question 2

What is a key factor in production of high quality cytogenetic harvests?

Answer 2

• correct prep of lab cell lines or cell samples obtained from patients
• cells should be cultured until approp no. present and users should take
  care to prevent over confluency in pop of cells

Question 3

What is the problem w/ over-confluency?

Answer 3

• depletion of culture media/increased environmental stress in confluent cultures causes alts to gene expression and biochemical profiles, which can cause abnormal experimental results
• also prevents cells from progressing t/ cell cycle and if performing analysis specifically of metaphase
  chromosomes, cells must be proliferating and have an active cell cycle at the time of
  harvest

Question 4

How is no. metaphases increased?

Answer 4

• colcemid treatment (2-4hr)
• at low concs, binds to tubulin proteins at microtubule plus ends,
  preventing polymerisation
• at higher concs, it promotes microtubule detachments from MTOCs, leads to depolymerisation of detached microtubules
• so cells cannot form
  mitotic spindle and initiate chromosomal seg during cytokinesis, so arrest in metaphase

Question 5

Why are cells swollen w/ hypotonic solution?

Answer 5

• genetic material tightly compacted w/in nucleus during normal cell division, making visualising and analysis of indiv chromosomes difficult
• hypotonic salt solution causes water to move into cell by osmosis and increase the vol of cytoplasm and nucleus
• use KCl

Question 6

Why is fixative req?

Answer 6

• stabilises cellular morphology, ceasing biochemical activity, inactivating cellular
  enzymes and preventing sample deterioration
• use Carnoy’s solution = methanol:acetic acid fixative added to cells immediately after incubated in a hypotonic solution

Question 7

What is the components of fixative, and their purpose?

Answer 7

• 3:1 methanol to acetic acid
• methanol dehydrates cells, precipitate proteins and permeabilises membranes, causing cell shrinkage, but reduces cell swelling
• acetic acid denatures some proteins and counteracts methanol-induced cell
  shrinking to maintain cells in a swollen state