Introduction.Hi.We are from the Institute of Biometrical Science at the Federal University of Faja in Brazil. In this video we'll be preparing chromosome spreads of human embryonic stem cells to detect the possible miracle chromosome alterations. Although human embryonic stem cells have been shown to present a stable deployed karyotype, men studies have reported that depending on keter conditions, they become prone to acquire chromosome anomalies such as addition of whole or parts of chromosomes.
The characterization of the genetic integrity of HASS in vitro is crucial considering as an essential two and embryogenesis studies and drug testing. Furthermore, for future therapeutic proposals, chromosomal changes are a real concern as it is frequently associated to carcinogenesis. So let's get started.
Treating the cells with ate In this procedure, we use it. The eight to nine has line cured in the F 12 supplemented with 20%of no count serum replacement and 80 to nine grams per milliliter of fibroblast growth factor. Begin by removing the cuter flask from the incubator and under a laminar flow hood, add coit that final concentration of 0.1 microgram per milliliter.
The addition of Coit to the median induces mitoka, rest and metaphase. When the chromosomes are well condensed and can be more easily visualized the under microscope, be sure to use the sterile techniques to avoid contamination. When have added coit kept the flask and returned the cells to the incubator.
Fixing the cells after the incubation period, the cells are not ready to be fixed at this point. It's not necessary to work under a sterile condition. Begin by aspirating the median and when you finish it, add a tryin.
The DTA is 0.05%to the flask. Be sure to add enough tryin sufficient To cover the bottom surface of the cells. Cap the flask and gently rocket To even distribute.
The trypsin allow cells to incubate for five minutes at Rome temperature. After the five minutes of incubation at Rome temperature, the cells should no longer be attached to the bottom side of the flask and you'll need to inactivate the trypsin either by adding cutter median containing serum or by gently adding serum to the cells. Carefully remove the cap of the flask and add cutter median or the serum, allowing the liquid to lie down the bottom side of the flask over the cells.
Pipette the cell suspension up and down several times to completely dissociate the cells. Transfer the cells to a 15 milliliter conical tube, but before doing so, make sure that the cells have been well dissociated and that the suspension doesn't contain any visual cell clump. Once you have completed the transfer cap the tube and centrif.
Fold the suspension for five minutes. At 800 RPM after centrifugation, there should be a cell palette at the bottom of the tube. Be careful to not disturb the cell palette.
Aspirate the sate using a P 1000 until there is only a approximately 200 microliters remaining. This is a volume median to Resus. Suspend the palate to Resus.
Suspend the palate. Flick the tube gently with your fingers Several times. Make sure that the cells have been well suspended by checking if there is no clump of cells remaining the solution.
At this point, the solution should be tur. Next, you need to add five milliliters of 75 millimolar solution of potassium chloride prewarm to 37 degrees Celsius. The hypotonic solution should be added very slowly by the tube below while you rotate the tube.
After you have added the first three milliliters. Cap the tube tightly and carefully. Homogenize the cell suspension with a hypotonic solution By gently turning the tube horizontally and is slowly routing.
It proceed by slowly adding the remaining two milliliters in the same manner as before. The proposal of the hypotonic solution is to increase the cell volume to allow the chromosomes to untangle. And so this incubation period is crucial for obtaining good chromosome spreads when the entire five milliliters have been added, cap the tube and incubate the cells in a water bath statutory seven degrees Celsius for 15 minutes.
When the 15 minutes incubation period is completed, remove the tube from the water bath. At this point, you may work on the fume hill that the fumes produced by the fixative solution are toxic. If you do not have access to a fume hood, be sure to work on a well ventilated area.
Remove the cap of the tube and add the three drops of the fixative solution. This is a three to one R of methanol and acetic acid and should be prepared on the day of use. Cap the tube tightly and gently turn the tube horizontally while it is slowly rotating it First clockwise, then counterclockwise.
Centrifuge the tube for five minutes at 800 RPM without break. It's very important that spin cycle is allowed to stop slowly to prevent loss of material caused by cell attrition After centrifugation. This cardio sate live in about 200.
Just to suspend the palate, flick the tube gently with our fingers several times. Make sure that the cells have Been well suspended by checking if there's no clump of cells remaining. The solution At the three milliliters of fixative solution is slowly by the tube wall while you rotate the tube after, have added the first three Milliliters.
Cap the tube T tightly and carefully homogenize the cell suspension by gently turning the tube horizontally and is slowly rotating it. Be sure to make at least one full rotation. To maximize this mixing process, add more.
Two milliliters of the fixative solution by the tub wall to take the cells down. Stock the cell suspension at four degrees Celsius overnight. This fix it cells can be stored in fixative solution at four degrees Celsius for months.
Washing the cells on the next day centrif for the cells for five minutes at 800 RP Without break. Discard to sate and live in about 200 microliters.Raise. Suspend the pellet by flicking the tube with Their fingers several times.
Make sure that the cells have been well associated and that the suspension doesn't contain any visual cell clump. Add five milliliters of fresh fixative solution slowly by the tube wall. Cap the tube and homogenize the cells by gently turning the tube horizontally while it slowly rotating it.
Besides protecting the cells in there will state the fixative solution, removes lipids and then aist proteins, and as a result, make the chromosome spreading easily. Centrif foods, the tube for five minutes at 800 RRP M without break. Preparing the slides At the centrifugation step leaves sufficient amount of solution to homogenize the cell palate.
This amount of liquid may depend on the number of cells. Flick the tube gently with their fingers several times. Make sure that the cells are well associated by checking if there is no visual cell clump.
At this point, the solution should be target added, 20 to 30 microliters of cell suspension onto a clean, dry slide by moving the pipette parallel to this light surface. As the fixative solution evaporates the surface of light becomes grainy immediately. Expose this light face up into this steam of hot water for 30 seconds to cause the cells blow up.
Analyzing these lights. After preparing these slides, analyze under phase contrast microscope using the 10 and the furry objectives to see if the cell density is suitable for further analysis. This slide is an example of one that is too concentrated.
Note that the cells nucle are too close to the metaphase spreads. If you have a slide where the cell density is too high, then the lut cell suspension by adding more fixative solution. This slide shows the desired cell density.
Note that the metaphase spreads are well dispersed, isolated. Once a slide with an optimal cell density has been obtained, it cannot be stained and analyze it. Avoid choosing the metaphase which are near nucleus as chromosomes may be hiding or block from view.
Look for metaphase which are rounded, but be sure that no chromosomes have straight away from the metaphase YO two choosing metaphase that are rounded and well isolated. As in the following slide, you try to avoid reporting false IES as the gain of chromosomes generated by two more spreads. Mix it together, or the loss of chromosomes generated by straight chromosomes.
We hope that this video will help you on your further experiments. If you have any doubt, do not hesitate to contact us. So enjoy yourself and good luck.
