Currently, I am trying to do karyotyping on HeLa cells. The protocol is as followed.
HeLa cell karyotyping protocols
1. Culture the HeLa cells in a 6 well-plate with 80% confluence.
2. Discard the medium and add fresh medium (DMEM, 10% FBS, 0.1% gentamicin ).
3. Add colcemid to the medium with final concentration of 0.06 ug/ml.
4. Incubate in 37°C, 5% CO2 for 4 hours.
5. Prewarm 0.075M KCl solution in 37°C.
6. Prepare Carnoy’s fixative solution (ratio of acetic acid to methanol 1:3) and store it in 4°C.
7. Discard the medium and wash the cells with PBS twice.
8. Add trypsin to detach the cells (3 min in 37°C) and place the cells solution in 15ml falcon.
9. Centrifuge to get the cell pellet (1000 rpm, 5 min).
10. Discard the supernatant and completely re-suspend the pellet with 200ul PBS.
11. Add 5ml 0.075M KCl solution by gently rotating the tube and flicking it.
12. Add another 5ml 0.075M KCl solution by gently rotating the tube and flicking it.
13. Incubate the cell solution in 37°C, 30 min. Add 1/10 fold of fixatives into the cell solution.
14. Centrifuge to get the cell pellet (1000 rpm, 10 min, without brake).
15. Discard the supernatant.
16. Add 5ml Carnoy’s fixative solution and re-suspend the pellet mild vortex.
17. Add another 5ml Carnoy’s fixative solution without vortex.
18. Centrifuge to get the cell pellet (1000 rpm, 10 min, without brake).
19. Discard the supernatant.
20. Add 5ml Carnoy’s fixative solution and re-suspend the pellet by mild vortex.
21. Repeat step 18-20 twice.
22. Discard the supernatant with 0.3ml solution kept in the tube.
23. After gently resuspending the pellet, pipette 3-9 drops of the cell suspension from a distance of about 30cm onto a slide which is tilted at an angle of about 45° and allow the suspension to roll across the slide. Add one large drop of fresh Carnoy's Fixative to the slide.
24. Observe the chromosome under microscope with power of 400X.
My question is that most nucleus are not burst to release the chromosome,
the length of chromosome is too short and there are not spread enough for further analysis.
Anyone can give me suggestion on that?
Dear Dr. Ihsan E Al-Saimary,
can you give me some suggestion based on my protocol?
to lengthen chromosomes - reduce the colcemid incubation time to 2-2.5 hours or reduce its concentration, e.g., 0.03 u/ml
to remove the cytoplasm - increase the time of incubation in hypotonic solution (KCl), e.g., 10-15 min
Hello,
After you drop the cell suspension on the slide, hold it at an angle for about 15 seconds and then put the slide on a hot plate at about 60 degree till dry.
In clinical, we used in situ culture for preparing the chromosome.
Hella cell is attached cell. You can culture the cells on the cover slip (22*22 mm)
Until the cells growth almost 50-60% (After you seed the cells on the slide, you have to wait for many 2-3 days then to go to harvest the cells)
4. Incubate in 37°C, 5% CO2 for 18 mins. (If your cells are all active cells, don’t worry about the division index to low.)
5. Add prehypotonic solution for 5 mins (Prewarm 0.28% KCl solution in 37°C)
6. Discard the solution then add the hypotonic solution for 3 ml in petri-dish contained coverslip for .15 mins in 37°C oven.
7.Add prefix (methanol: acetic acid=3:1) for 5 mins, Discard the solution then add 3 ml of fix solution in petri-dish contained coverslip for .10 mins. Repeat 3 times for this step
8. use 45 °C heating to break the cells and spread the chromosomes. (cover slip would stick on the slide)
K..S, Lavappa, a post-doc of mine, was in the process of clarifying the massive human cell contamination that had accumulated at the ATCC by the overwhelming HeLa cell contamination that occurred in a number of labs in the course of establishing neoplastic cell lines from human neoplasms. Prior to his unfortunate death, he had amassed sufficient evidence following specific marker chromosomes of HeLa to demonstrate that all human neoplastic cell lines at the time were contaminants of HeLa.
Lavappa's karyotyping was truly superb. While presenting his findings at the annual meeting of the Tissue Culture Association, the audience spontaneously burst to a lingering applause as he displayed truly superb Giemsa stained karyotypes displaying HeLa's marker chromosomes in all cell lines preserved at the ATCC.
Earlier, Stanley Gartler caused bedlam at an earlier TCA meeting (Seattle?) when he clearly demonstrated, using the single and double banded features of the LDH enzyme that distinquishes the origin of cells derived from whites vs.blacks, respectively, by the single vs. double banded nature exhibited solely by HeLa cells. All so-called human neoplasms cryopreserved at the ATCC had the characteristic double-band that is solely exhibited by tissues of black origin. Once Gartler stopped his presentation, all :"hell" broke loose in the conference room as individuals who derived the alternate cell lines rose up to defend their findings. It was truly a moment to remember.
I suggest you look up K.S. Lavappa's publications to note if any of his technical steps may be helpful.
HeLa contamination was first reported by Ford and myself in 1958 at the time the Chinese hamster V79 cell line was derived. Gartler's publication is in Nature 217: 750, 1968.
George Yerganian ([email protected])
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