The overall goal of this procedure is to harvest and prepare chromosomes for GB banding and molecular cytogenetic tests. First, culture the cells to logarithmic phase. Then harvest the chromosomes with smid, a hypotonic and a fixative.
Drop the cell suspension into slides. Stain one slide. As a monitor of the chromosomal preparation, proceed to GB banding or molecular techniques of the remaining slides.
As a final step in GB banding, analyze the chromosomes by light microscopy and karyotyping. Ultimately, the chromosomes and nuclei will be available for karyotyping or fish ish. I first had the idea for publishing this method because of the high demand from other researchers requesting assistance in the detection of chromosome instability in different types of cells from various organisms and for subsequent molecular cytogenetic testing.
This technique is essential in the diagnosis and ultimately prognosis of many congenital genetic disorders including cancer, because it allows for the visualization of chromosome rearrangements, which are diagnostic. For these disorders, Though this method can provide insight into the chromosome rearrangements found in various types of human cells. It can also be applied to cells from other organisms such as Resus Maca rats and mice Culture.
2 million adherent cells to 80%con fluency and add 10 microliters of 10 micrograms per milliliter. Col per milliliter of cells. Incubate the cells at 37 degrees Celsius in a 5%CO2 incubator for 45 minutes.
Then using a sterile pipette, transfer the media from the culture into a 15 milliliter conical tube and reserve for later To wash the cells, add two milliliters of HBSS into the flask, swirl and remove the buffer. Next, add one milliliter of trypsin, ensuring it covers the entire surface of the flask. Once the majority of the cells have detached, pipette the media in the conical tube back onto the cells.
Aliquot 10 milliliters of the cell suspension into 15 milliliter conical tubes. Centrifuge at 200 Gs for 10 minutes. Remove the supernat.
Re suspend the pellet in 10 milliliters of 75 millimolars potassium chloride, which will be prewarm to 37 degrees Celsius after a 10 minute incubation of 37 degrees Celsius. Centrifuge at 200 Gs for five minutes. At 25 degrees Celsius, remove all but 0.5 milliliters of the supernatant and resuspend the pellet Carefully.
Add five milliliters of fresh car noise fixative to the cells. Then add five milliliters, more of the fixative and vortex centrifuge at 200 Gs for five minutes. Resus, suspend each cell pellet in five milliliters of fixative.
Store the cells at four degrees for up to one year. Centrifuge the cells at 200 Gs for five minutes. At 25 degrees Celsius.
Remove the sup natant until only 0.3 to 0.5 milliliters remains after Gently resus suspending the pellet. Pipette three drops of the cell suspension from a distance of about two inches onto a slide which is tilted at an angle of about 45 degrees. And allow the suspension to roll across the slide.
Add one large drop of fresh car noise, fixative to the slide. Drive the back of the slide on a paper towel, and then sit the slide out to dry completely. Prepare fresh gim sustaining solution.
Place the slides on a staining rack. Cover the entire slide in the gim sustaining solution. After five minutes, rinse the slides with distilled water drain and allow to air dry.
Add four drops of perm mount to the slide and place a cover slip ensuring there are no bubbles under the cover slip. Remove the excess per mount with a paper towel. Analyze the cells with a light microscope under 10 x and 100 x magnification.
If the metaphase cells are abundant and well spread, the remaining slides can be used for other experimental procedures. Add 50 milliliters of the treatment solutions to four Copeland jars. Immerse each slide in jar number one for five seconds after.
Quickly rinsing the slide in jar number two. Leave it in jar number three for at least 30 seconds. Rinse in jar number four, then allow the slides to dry.
Prepare fresh GIM sustaining solution. Place the slides on a staining rack. Cover the entire slide in the gim sustaining solution for five minutes.
Rinse the slides and distilled water in the same order that they were stained. Then dry for at least 10 minutes. Place a cover slip using perm mount as shown earlier.
Then analyze the cells under a light microscope for the remaining slides. Adjust the tripsin incubation time. Based on the results of the first slide.
A successful assay yields chromosomes that are well spread and of suitable chromosome morphology.Properly. GB banded chromosomes contain the characteristic light and dark banding patterns. After watching this video, you should have a good understanding of how to harvest chromosomes for GB banding and further molecular cytogenetic testing in the elucidation of chromosome instability.
Once mastered, this harvesting technique can be done in about three hours if it is performed properly. For the GB banding procedure, it's important to initially test the tripsin incubation time on one slide before proceeding to GB band. The rest of the slides.
