Overview
- Preparation of metaphase chromosome spread is an important technique in cytogenetics. Metaphase chromosome spread is used for karyotyping including analysis of numerical or structural changes in chromosomes.
- Prepared metaphase chromosome spread can be further analyzed by several techniques including G-banding, D-banding, in situ hybridization, SKY, etc.
- Metaphase is one of the stages of mitosis which is characterized by the appearance of chromosomes due to condensation of duplicated genetic material (chromatin/genomic DNA). At the end of metaphase, chromosomes are aligned at the metaphase plate. In the next stage of mitosis, the anaphase, sister chromatids are separated and dragged to opposite poles with the help of mitotic spindles.
- Colcemid, an inhibitor of the mitotic spindle, inhibits the formation of mitotic spindles, thus stopping the segregation of chromosomes and cells remain in the metaphase.
- These metaphase cells are lysed in a controlled manner on a slide that causes the release and spread of chromosomes from individual cells that remain close to each other, thus making it possible to analyze chromosomes from individual cells.
- To lyse metaphase cells in a controlled manner, cells are treated with a hypotonic solution that results in an increase in the volume of cells and cells become prone to lyse upon mechanical stress. Cells are fixed with Carnoy’s fixative and are dropped from the height onto a slide. Cells burst when they hit the slide surface, resulting in the release of chromosomes due to lack of nuclear membrane in metaphase cells. By controlling the cell density in suspension, a chromosome spread of individual cells can be obtained.
Requirements
Reagents and solutions
♦ Exponentially growing cells (70 % confluent) in T75 tissue culture flask
♦ Colcemid stock solution (1 µg/ml in PBS, Catalog number: 15212012, ThermoFisher Scientific)
♦ 75 mM KCl solution
♦ Trypsin-EDTA solution
♦ Carnoy’s fixative (3:1 methanol: glacial acetic acid) (prepare freshly)
Equipment and disposables
♦ Pasteur pipette
♦ Micropipettes and tips
♦ Clean glass slide
♦ Centrifuge tubes (15 ml)
♦ Centrifuge
♦ Gloves
Objective:
Preparation of metaphase chromosome spreads from adherent cell line
This is a general protocol for the preparation of metaphase chromosomes spread from any adherent cell lines.
Starting material:
Exponentially growing adherent cells (70 – 80% confluent T75 tissue culture flask)
Note:
Make sure cells are healthy and are not in the stationary phase of the growth curve. It is recommended to change the culture medium a day before you start the experiment.
Protocol
Step 1: Colcemid treatment to adherent cells
♦ Add colcemid stock solution (10 µg/ml) to a final concentration of 0.1 µg/ml to the culture medium of the flask. You need to add 100 µl colcemid stock solution to 10 ml culture medium to get a final concentration of 0.1 µg/ml.
♦ Incubate cells in standard culture condition (e.g., at 37 °C in a CO2 incubator for mammalian cell lines growing in DMEM medium) for 1 to 2 hours in the cell culture incubator.
Note:
One must standardize the incubation time for each cell line. Past experiences or literature search can help to standardize the colcemid treatment time. Generally, for fast-dividing cells, 45 min – 1 hour and for slow dividing cells 2 hours treatment time is sufficient. Longer incubation with colcemid can compromise the quality of chromosomes whereas less treatment time results in a low number of mitotic cells.
Tips:
Since the direct mixing of colcemid with the culture medium of the flask can cause cell detachment, the easiest way is to take out half of the culture medium from the flask in a 15 cm tube and add 2 x concentration (i.e., 100 µl of stock solution) of colcemid. Mix by pipetting and transfer this medium back to the culture flask. Agitate the flask gently to mix it.
Precaution:
Don’t replace the flask culture medium with a fresh medium containing colcemid. A fresh medium can slow down the cell cycle progression.
Step 2: Harvesting colcemid-treated adherent cells from the flask using a standard subculturing procedure
♦ Remove the medium from the flask and wash the cells gently with PBS (10 ml PBS for a T75 flask).
♦ Add sufficient amounts (2 ml for a T75 flask) of trypsin-EDTA (or any suitable cell dissociating agent for your particular cell line) and spread all over the flask by tilting the flask in all directions. Incubate for 1 – 2 min in cell culture incubator.
♦ Once the majority of cells are detached, add 10 ml of serum-supplemented medium to stop the trypsin action. Pipette medium up and down a few times to make a single-cell suspension.
♦ Transfer cell suspension into a 15 ml centrifuge tube and harvest cells by centrifugation (200 g for 5 min).
Note:
Colcemid arrests cells in mitosis. Mitotic cells attach loosely to the substratum. Therefore, there is a risk of losing these cells while removing the culture medium and during the washing step with PBS. In such cases, you can also recover cells from the culture medium and PBS wash by centrifugation.
Step 3: Hypotonic treatment to cells
♦ Pour out the supernatant. There will be some remaining medium (approximately ≈50 µl) in the centrifuge tube. Resuspend the cells by tapping or vortexing the tube briefly.
♦ Add 10 ml 75 mM KCl while tapping the tube very gently. KCl should be added slowly from the side of the tube using Pasteur pipette or 1 ml pipette.
♦ Incubate for 8 – 15 min.
Precaution:
The hypotonic treatment causes an increase in the volume of cells therefore, cells are susceptible to burst even due to mild mechanical stress.
Step 4: Fixation of cells
♦ Add 5 ml freshly prepared fixative solution (3:1 Methanol:Glacial acetic acid) slowly and gently from the sidewall of the tube. Mix by inverting the tube gently.
♦ Wait for 5 min and harvest cells by centrifugation (200 g, 5 min)
♦ Pour out the supernatant and resuspend the pellet in the remaining liquid by tapping the tube.
Tip:
Fixative should be added dropwise from the sides of the tube with constant gentle agitation.
Precaution:
1. Don’t centrifuge to collect the cells before fixing the cells. Cells may form insoluble clumps if centrifuged without fixation.
2. Do not remove the supernatant completely to avoid drying of cell pellet. Moreover, this leftover amount of supernatant can be used to resuspend the pellet by tapping the tube.
Step 5: washing cells with fixative 3 – 4 times
♦ Tap the tube to resuspend the cell pellet gently. Add 5 ml Carnoy’s Fixative and make uniform suspension of cells by inverting the tube 3 – 4 times. Harvest the cells by centrifugation (200 x g for 5 min).
♦ Repeat this step 3 – 4 times.
Fixed cells can be stored at 4 °C for up to 1 week and at -20 °C for 6 months.
Wash stored cells 3 -4 times with fixative before going to the next step.
Step 5: Preparation of chromosome spread
♦ Resuspend the cells in 200 ml of fixative solution.
♦ Rinse the clean glass slide with fixative, air-dry it.
♦ Place the slide in the horizontal position.
♦ Drop cell suspension using a pasture pipette or pipetman (p100/p200) onto the slide from a distance (about 50 – 100 cm). Few (2 – 3) drops per slide is good for inspection of metaphase spread quality.
♦ Air-dry the slide and analyze it under a microscope.
