This protocol can be used for assessing attachments to the nucleoskeleton rather than using DamID sequencing, and it's a single cell assay. The main advantage of using this method is that it's cost effective and easily accessible. It can highlight functional differences between genes and chromosomes that may not normally occur with other visualization techniques.
Spatial and functional organizations within the genome in treated cancer and progeria cells and genomic behavioral differences in senescent cells can be assessed using this protocol. This method can be applied to any cell or tissue type across the lifespan and of any disease. It can also be used in model organisms where probes are available.
To begin, prepare 500 milliliters of 10%hydrochloric acid and pour it into a large beaker. Drop microscope slides individually into the acid and incubate them for one hour at room temperature on a shaker set to two G.Decant the acid from the beaker and wash the slides 10 times in tap water. Then a further 10 times in deionized water.
Rinse the slides in methanol twice and keep them in methanol until sterilization. Tip the slides onto a paper towel to dry and wrap them in foil for sterilization in an autoclave or hot oven. Grow cells in the appropriate medium with serum for at least 48 hours at 37 degrees Celsius and 5%carbon dioxide until 60 to 70%confluency is reached.
Harvest each cell type and count the cells using a hemocytometer. Seed one times 10 to the five cells in 10 milliliters of medium per slide. Remove the culture dish containing the slides and attached cells from the incubator.
Discard the medium and label the slides using a pencil. Then place them in a coplin jar containing 50 milliliters of ice cold cytoskeleton buffer. Incubate the jar for 15 minutes on ice or at four degrees Celsius.
Discard the cytoskeleton buffer and quickly rinse the slides in 50 milliliters of one-time DNA halo buffer three times by dipping them into a coplin jar with the buffer. Transfer slides to a coplin jar containing 50 milliliters of extraction buffer and incubate for four minutes at room temperature. Then consecutively incubate the slides in 50 milliliters of 10 times, five times, two times, and one time DNA halo buffer for one minute each.
When finished, dip the slides through a sequential ethanol series of 10, 30, 70, and 95%ethanol. Take the slides through a sequential 50 milliliter ethanol series of 70, 90, and 100%ethanol for five minutes each. Air dry them on a warming plate.
Then bake in a 70 degree Celsius oven for five minutes. Denature the slides by placing them in 70%formamide two times SSC solution for two minutes at 70 degrees Celsius. Place the denatured slides in 50 milliliters of ice cold 70%ethanol for five minutes.
Then move them through an ethanol series of 90, 95, and 100%at room temperature for five minutes each. When finished, air dry the slides on a warming plate. Denature the DNA probes at 75 degrees Celsius for 10 minutes in a hot block or water bath.
Then incubate them at 37 degrees Celsius for 30 minutes before pipetting 10 microliters onto the appropriate slide. Overlay the probe with a 21 by 21 millimeter coverslip and seal it with rubber cement. Incubate the slides for a minimum of 18 hours at 37 degrees Celsius in a humidified hybridization chamber.
After the incubation, carefully remove the rubber cement with forceps. Then incubate the slides in 50 milliliters of 50%formamide two times SSC solution at pH seven that has been pre-warmed to 45 degrees Celsius for three five-minute incubations. Next, place the slides in 50 milliliters of 0.1 times SSC solution that has been preheated to 60 degrees Celsius, but placed in a 45 degrees Celsius water bath.
Incubate for five minutes and replace the buffer twice for five-minute incubations. Place the slides into a coplin jar containing 50 milliliters of four times SSC solution at room temperature and incubate for 15 minutes with three changes of buffer. To prevent non-specific antibody binding, apply 100 microliters of 4%BSA four times SSC solution to each slide and overlay it with a piece of paraffin film.
Incubate at room temperature for 10 minutes. Then incubate the slides with 100 microliters of streptavidin Cy3 diluted one in 200 for one hour at room temperature to detect the labeled probe. Place the slides into a coplin jar containing 50 milliliters of four times SSC solution at room temperature and incubate for 15 minutes with three changes of buffer.
Mount the slides in 20 microliters of a mounting solution containing DAPI and overlay with a 22 by 50 millimeter coverslip. Visualize DNA halos and chromosome territories using an epifluorescence microscope with a 100X oil objective. DNA halo preparation makes it possible to see the edge of a residual nucleus, the DNA remaining within the residual nucleus, and the unattached DNA that has spooled out into the surrounding area.
DNA halos of proliferating human dermal fibroblasts were created from cells with incorporated BRDU and subsequently stained with anti-BRDU antibody. Proliferating cells were positive for BRDU and anti-pKi-67. This protocol was used to visualize chromosome territories within DNA halos.
Individual chromosomes were labeled with specific whole arm chromosome painting probes for chromosomes 1, 13, 17, and 18. Anti-pKi-67 was used to mark proliferating cells and its absence within the same culture. DNA halo preparations with telomeres in green are shown here.
It's possible to observe the proportion of telomeres in the DNA halo, particularly in the experiment two image. The mean percentage of telomeres was approximately 17%in quiescent cells. Differences in chromosome attachment were detected in primary control fibroblasts and in diseased cells with typical and atypical Hutchinson-Gilford progeria syndrome, expressing a different Sun1 isoform and no Lamin A mutation.
Chromosomes one and 13 show statistically significant differences in their attachment within the residual nuclei when compared to controlled DNA halos. The position of the whole chromosome territory was correlated to the residual nucleus and DNA halo. When attempting this protocol, make sure that cells are seeded at the correct density and extracted at the right time.
Following this procedure, indirect immunofluorescence and RNA FISH can be performed.
