Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence

Our research primarily focuses on the connection between DNA repair and aging. Specifically, we look at different laminopathy disorders and we want to learn how they may impact DNA repair. We have demonstrated that a laminopathy disorder associated with premature aging and a short lifespan known as Hutchinson-Gilford progeria syndrome is actually associated with an increased amount of DNA damage and imprecise DNA repair.

Our lab would like to investigate the effect of potential therapies for laminopathy patients to learn if these approaches will also have a positive impact on DNA repair and DNA damage. To begin, place one cover slip into each well of a sterile 6-well plate using forceps. Add two milliliters of supplemented Dulbecco's Modified Eagle Medium, or DMEM, to each well in the 6-well plate.

Aspirate the media from the HeLa cells cultured in the flask and wash the cells by adding 15 milliliters of PBS. Swirl the flask gently to wash the cells, aspirate the PBS, and add two milliliters of trypsin-EDTA to coat the cells. Place the flask coated with trypsin in a 37 degree Celsius and 5%carbon dioxide humidified incubator for two minutes to allow cells to detach.

Then add eight milliliters of supplemented DMEM to the flask containing the trypsinized cells and pipette up and down several times to separate clumping cells. Collect the cell suspension from the flask into a 15-milliliter polystyrene tube. After counting the cells, add 500, 000 cells drop by drop directly onto each of the cover slips in the 6-well plate.

Place the plate into a 37 degrees Celsius, 5%carbon dioxide humidified incubator to allow the cells to grow overnight. For cell fixation, aspirate the media from the wells of the 6-well plate the next day. Add two milliliters of PBS to each well to wash the cells and aspirate all PBS completely from the wells.

Then add two milliliters of 4%formaldehyde solution to each well for 10 minutes at room temperature and aspirate the solution completely from the wells. Next, add two milliliters of permeabilization solution to each well. Allow the solution to sit for 10 minutes at room temperature, then aspirate it completely.

Wash the cells three times with two milliliters of PBS in each well. Aspirate fully after each wash. Once all PBS is aspirated from the wells, add two milliliters of antibody diluent buffer, or ADB, to each well to block the cells for immunofluorescence staining.

Incubate at room temperature for 30 minutes with gentle orbital shaking. Towards the end of the blocking period. Prepare the primary antibody dilution by mixing lumen B1 and double-stranded DNA antibodies in ADB.

At the end of the incubation, aspirate the ADB from the wells. Tape down a piece of paraffin film on a flat surface, ensuring the area is large enough to accommodate all the cover slips being processed. For each cover slip, pipette 75 microliters of the primary antibody dilution onto the paraffin film avoiding any bubbles.

Using forceps, place each cover slip cell side down onto the antibody droplet. Cover the incubating cover slips with the lid of the 6-well plate and allow the cover slips to incubate for one hour at room temperature. Then using forceps, carefully remove the cover slips from the paraffin film and place them back into the 6-well plate with the cell side facing upward.

Wash the cover slips by shaking them three times for five minutes with two milliliters of PBST. During the final wash, prepare the secondary antibody dilution. After the last wash, aspirate PBST completely.

Add secondary antibody to the para film and place the cover slips on paraffin film, cell side down. Then place a light protective cover over the cover slips to protect the cells from light and incubate for 30 minutes at room temperature. Using forceps, carefully remove the cover slips from the paraffin film and place them back into the 6-well plate, ensuring the cell side is facing upward, Then dehydrate the cover slips by sequentially adding two milliliters each of 70%90%and 100%ethanol to the wells, allowing each solution to sit for one to two minutes before removal.

Using forceps, remove the cover slips from the wells and place them on a lint-free wipe to air dry, protected from light. Mount the cover slips cell side down onto glass microscope slides using 20 microliters of mounting medium per cover slip. Nuclear blebbing was more prevalent in HeLa ZMPST24 knockout cells with approximately 50%of nuclei exhibiting blebs, compared to 17%in HeLa control cells.

DNA leakage was observed predominantly in HeLa ZMPSTE24 knockout cells, while no DNA leakage was observed in HeLa control cells, DNA leakage occurred in some ZMPSTE24 knockout cells, even in the absence of visible nuclear blebbing.