I'm doing karyotyping in mouse embryonic stem cells,but it is really difficult to count chromosome number accurately, requiring a long time to find good metaphases.
I would like to know if someone could help me with a better way to spread chromosomes, and provide some tips to facilitate counting.
Thanks a lot!
Try to use use newly prepared good quality hypotonic. May be help you..
Hello
To an exponentially growing ES cell culture that has been fed at least one hour previously, add new media containing colcemid (Demecolcine - Sigma D-7385) to a final concentration of 0.02 mg/ml. (Stock of 2 mg/ml in water). Return to the incubator for 1 hour.
Start with a 6 cm dish (6 well also works ok). Probably don’t need that many cells but you lose a lot in the washes.
Prepare slides: Wash slides in fix (3:1 methanol : acetic acid) and then soak them in ice cold water until ready to use (distilled water plus some ice). It is important for the slides to be both cold and wet when ready to go.
Wash cells 2x with PBS. Trypsinize cells for 5 minues as usual. Add DMEM plus serum and resuspend the cells aiming for a single cell suspension. Spin down at 1000 rpm for 3 minutes. Remove supernatant.
Add 1 ml 0.56% KCl dropwise. Flick the tube to resuspend the pellet again aiming for a single cell suspension (no big chucks). Do not pipette up and down or all the cells will stick to the inside of your pipette. Add 4 more mls of 0.56% KCl. Incubate at RT for 6 minutes. This number is important as you are now swelling the cells.
Spin down the cells at 500 rpm for 5 minutes. Remove sup. Add 1 ml fix (3:1 methanol:acetic acid). Flick tube to resuspend pellet, add 4 more mls fix. Incubate 5 mins RT.
Repeat step #5 three more times. Finish with 1 ml total volume (or 0.5 mls if starting with 6 well dish).
Remove slide from the water, blot edges to remove excess liquid and drop the cell suspension (dropwise) from at least one foot above the surface of the slide (2-3 drops/slide). If the cells are dropped from too close a distance, they will not burst. So, make sure you drop them from high enough. One way to do it is to place slide on the floor and then drop the cells onto them from appropriate height). Allow the slides to dry (can blow on them or place them under heat lamp to speed up the drying).
Prepare 3 or 4 slides for each sample. The cells can be stored at -20C but rumor has it that the slides are best prepared the same day.
Stain slides with Giemsa (1:20 dilution) (Giemsa Modified Stain - Sigma GS-500) for at least 15 minutes (can go longer... even overnight). Wash slides with water.
Photograph chromosome spreads to count. Do not try to count under the microscope as it will not be accurate.
this protocol is modification of Dr. Patricia Labosky's protocol. Courtesy of Raluca Verona from the laboratory of Dr. Marisa Bartolomei, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine
Ref ( https://www.med.upenn.edu/ )
Good Luck
Hi
I am just wondering whether your technical problem with karyotyping of mESCs solved.
I know very good and expert person in Royan institute , Her name is Najmeh Massoudi. If you can contact her, she is one of the best and helpful person in Royan Institute.
Good luck,
As Saleh said it helps adding colcemid or colchine, but normaly with mESC growing at their normal rate you should get plenty metaphases. In my hands the critical steps was the height and even more aiming properly the drop on the coverslip. use the position of your hand in respect to your head as reference. you will need a couple of tries to get the proper one, then measure it andn write it down.
another trick, put the coverslips on dry ice to ensure they're really cold.
For the counting you have to be really patient and be realistic with the acceptable n, I was doing fusion between ESC and SSC and we realised that at least 15 are a must but between 20 and 40 should do the job.
Finally check if you have a medical faculty or hospital around, if big enough they have Karyotyping facilities and setting the software to count mouse chromosomes won't be an issue. They may ask you, though, to have the cells ready and fixed.
Good luck