The aim of this procedure is to label chromosomes with paint probes to identify chromosome aberrations and segregation defects in mice and humans with polycystic kidney disease. This is accomplished by first harvesting cells from humans on mice and treating with smid, followed by fixing and preparing metaphase spreads on glass slides. Next, the slide is pretreated to remove extra RNA and cytoplasm which might interfere with the hybridization.
This is done by digestion with RNAs and pepsin respectively. Then chromosome and probe denaturation is performed followed by hybridization to label chromosomes. The final step is detection of the probe with fluorescent dyes.
Ultimately, results can be obtained that show chromosome numeral and structural abnormalities through fluorescence, microscopy and image capturing and analysis by Skyview software. The main advantage of this technique over existing methods like classical GIMs bending techniques, is that it provides additional information about complex chromosomal aberrations like chromosomal translocations, and complex chromosomal rearrangement that cannot be characterized by classical bending techniques. This method can help answer key questions in the polycystic kidney disease field, such as the mechanism of cystic kidney formation.
Demonstrating the procedure will be Dr.Wiam Abu, allowing a talented senior postdoc from our laboratory To begin al endothelial cells In DMEM containing 10 to 15%fetal bovine serum and 1%penicillin streptomycin at 37 degrees Celsius and 5%carbon dioxide until the cells reach 70 to 80%confluence. Then treat the cells with Cole solution at a final concentration of 0.05 milligrams per milliliter for 30 to 60 minutes at 37 degrees Celsius before harvesting to harvest the cells first, collect the medium containing any floating cells in 50 milliliters. Sterile fo centrifuge tubes.
Rinse the cells on the plate with sterile one times PBS after incubating with sterile trips in for one to two minutes, harvest and collect the remaining cells into the same tube. After pelleting the cells by centrifugation at 1000 rotations per minute for five minutes, aspirate the majority of the sate from the tube leaving around 0.5 milliliters on top of the pellet. Loosen the cell pellet by flicking the tube.
Next, depending on the pellet size, add five to 10 milliliters of a hypertonic solution of 0.56%potassium chloride in distilled water and incubate the suspension at 37 degrees Celsius for 30 to 45 minutes. Following the incubation, use a small one milliliter pipette to add one drop of an ice cold fixative solution per one milliliter of hypertonic cell suspension. Then invert the tube gently to mix after centrifuging the cells for five minutes at a speed of 1, 200 rotations per minute.
Aspirate the supinate as before. Next, slowly add one milliliter of fresh fixative solution while flicking the pellet. Then gently add another four milliliters of fixative to the wall of the tube.
This procedure is critical to ensure that the metaphase spreads will not be trapped in the cell clumps. After repeating the centrifugation step and aspirating the supernatant, replace the five to 10 milliliters of fixative by slowly pipetting the fixative down the walls of the tube. At this stage, the cells can be stored in a tightened and sealed tube at minus 20 degrees Celsius for short term or minus 80 degrees Celsius for long-term storage until ready to proceed.
When ready to proceed, check the number of metaphase chromosomes in the cell preparation. Clean the slide in absolute ethanol. Then tip the slide in distilled water approximately 10 times in order to form a sheath of water on the surface of the slide, place the slide on a glass plate and drop 15 to 20 microliters of cell suspension from 10 inches above the slide.
Place the slide in a water bath set at 65 to 70 degrees Celsius for one to two minutes and allow to dry check the slide under a light microscope using the 10 times and 40 times dry objectives. Make sure there are metaphase chromosomes and that the spreads are evenly spaced. Check the cytoplasm surrounding the chromosomes.
If cytoplasm is present as seen here, proceed with slide pretreatment. If no cytoplasm is present and the chromosomes have good morphology, then there is no need for slide pretreatment at 120 microliters of a freshly prepared one to 200 solution of 20 milligrams per milliliter. RNA solution dissolved in two times SSC to a 24 millimeter by 60 millimeter cover glass.
Invert the metaphase slide face down on the cover glass. Then gently invert the metaphase slide face up. Place the slide inside a humidified chamber and incubate it 37 degrees Celsius for 45 minutes.
Once the incubation time has elapsed carefully remove the cover glass without scratching the slide and washing two times SSC buffer in a coupling jar three times for five minutes each with shaking. Meanwhile, add five to 15 microliters of 100 milligrams per milliliter pepin in distilled water to a clean beaker, and then add 100 milliliters of 0.01 molar hydrochloric acid prewarm to 37 degrees Celsius. It is important to add the PEPs into the clean beaker first, or it'll not dissolve.
Transfer this solution to a coplan jar. Incubate the wash slide in the coupling jar at 37 degrees Celsius for no longer than three to five minutes after digestion. Wash the slide twice in a copin jar containing one XPBS for five minutes each with shaking.
Follow this with a five minute wash in a copin jar containing PBS supplemented with magnesium chloride. And then a 10 minutes incubation in a copin jar containing 1%formaldehyde, followed by a final wash in a cochin jar containing one XPBS for five minutes. Then dehydrate the sliding cochin jars containing a series of ethanol of 70%80%and 100%for two minutes each, and then air dry.
Observe the slide under a light microscope using a 40 times dry lens to ensure that the slides are digested properly, that no cytoplasm is present, and that the chromosome morphology is preserved. Finally, select an area for hybridization using a diamond pen. After freshly preparing the denaturing solution, place it in a copin jar and prewarm it to 70 degrees Celsius in a water bath.
Then place the slide in the cochin jar containing the Prewarm denaturing solution in a water bath at 70 degrees Celsius for 30 seconds to one minute. Following the incubation immediately place the slide in ice cold, 70%ethanol for three minutes, followed by 80%and 100%ethanol for three minutes each and then air dry. Examine the slide for chromosome morphology, the appearance of dark chromosomes that do not appear face light or shiny.
Denotes good chromosome or morphology. Warm the sky paint. Probe at 37 degrees Celsius with shaking for 20 minutes.
Then vortex and centrifuge briefly at 1000 rotations for minutes. For a few seconds, denature the probe in a thermocycler at 85 degrees Celsius for five minutes, followed by incubation at 37 degrees Celsius. For one hour, apply 10 microliters of the denatured probe onto the area of hybridization and cover with a 22 millimeter by 40 millimeter cover glass.
Being sure not to trap air bubbles seal the edges of the cover glass with rubber cement, then incubate in a humidified chamber at 37 degrees Celsius for 48 to 72 hours. Remove the cover glass carefully and place the slide in a coupling jar containing freshly prepared washing solution, one that has been pre-war to 45 degrees Celsius. Wash for five minutes, three times at 45 degrees Celsius in a shaking water bath at 45 rotations per minute.
Then wash the slides twice in washing solution two at 45 degrees Celsius for five minutes each with shaking, followed by five minutes in washing solution three of 45 degrees Celsius with shaking. Next, apply 80 microliters of blocking reagent. Then place a cover glass and incubator 37 degrees Celsius for 30 minutes.
Remove the cover glass and allow the fluid to drain. Apply 80 microliters of sci-fi staining reagent. Apply a cover glass and incubator 37 degrees Celsius for 40 minutes After the incubation, wash the slide with washing solution three of 45 degrees Celsius for five minutes, three times with shaking.
Next, apply 80 microliters of PS 5.5 staining reagent. Place a cover glass and incubator 37 degrees Celsius for 40 minutes after the incubation wash three times with washing solution three as before. Finally, tilt the slide and allow the fluid to drain.
Apply 20 microliters of the Antifa dappy reagent and place a 24 millimeter by 60 millimeter microscope. Cover glass carefully remove air bubbles that might have formed slides can be imaged immediately or stored at four degrees Celsius in the dark for no longer than one week. This sky image of a normal metaphase spread from a wild type mouse was captured using an Olympus microscope equipped with a 60 times oil immersion lens.
A custom designed triple band pass filter known as a spectral cube, a dappy filter, and a sac interferometer module with a CCD camera. Spectral karyotypes were carried out using skyview software. The top left panel shows the labeled metaphase spread, and the top right panel shows a brightfield image of the spread.
The bottom panel shows the chromosome sorted into ascending order using skyview software. This sky image of a metaphase spread from a pkd one homozygous knockout mouse shows abnormal chromosome number where 68 chromosomes are present instead of 40. Also visible are chromosome abnormalities such as the deletion of chromosome eight and chromosome or translocations, such as those between chromosomes five and 16, and also between chromosomes 11 and 19.
This sky image of a metaphase spread of human chromosomes from the vascular tissue of a non A-D-P-K-D patient has the normal female karyotype of 44 autosomes and two XXX chromosomes. This final sky image shows a metaphase spread from the vascular tissue of an A-D-P-K-D patient with an abnormal karyotype of 88 autosomes and four XXXX sex chromosomes. One master.
This technique can be done in five days if it is performed properly.
