Hello Dear Anissa,

To be very precise, grow primary culture of  cells from cancerous tissue (either by explant method or collagenase digestion), upscale the cells, then at 3-4th passage, arrest the cell division a methaphase by treatment with colchicine, put the cells in hypotonic soltuion so that cells will swell and now pour the cells on slide drop by drop.. Cells wil; burst and chromosomes will spread on slide. Now you can fix them and do giemsa stain and observe under microscope.

Hope it helps !

@Ajay Kumar thank you for the answer but I want some details in the protocol please :)

Hi

The chromosome preparation from blood / bone marrow / lymph node are text-book knowledge now, and if you are working with solid tissue [human?] it will be good to know the details of source to share the most relevant and workable protocol.  Ajaykumar has given perfect theoretical outline for how to go about it. It will be very helpful if one can spend some time in a lab where this is done to increase the success rate. Best regards

Dear Dr Nini

Thank you for this important question. As I am cancer cytigenetist and I have been working with cancer chromosome changes for 53 years, I have found no optimum protocol for chromosome preparation from fresh tumour sample. Even with the same tumour type there are so much variations in how to obtain chromosomes. Sarcomas need special treatment with collagenase to obtain free tumour cells while melanomas are soft and fragile and need special requirements. Carcinomas are so heterogenous as some of them are hard and others are soft and every type needs special treatment. You have to practice well then you would find and establish your own procedure. You may have a look for my paper i( 1.    Yaseen ,  N., Y.,  Watermore ,A,E,Potter,A .,M., Potter, C.W.,   Jacob.G, and Rees   R ,C. (1990) . chromosome studies in eleven colorectal tumours. Cancer Genet.  Cytogenet .44:83 -97). This may help you.

Regards

Dear Nini,

 I'm a cytogeneticist and working in a routine chromosome lab. We'r performing chromosome analyses from different tissues like: Amnion, Chorionic villi, Skin (Fibroblast cells), Blood and Bonemarrow. Unfortunaltely we do not work with solid tumours and therefore I dont have a proper protocoll for you. You could try to start with a standart protocol for chromosome preparation from fibroblasts.

But in "The AGT Cytogenetics Laboratory Manual",1997, Chapter 8: Cytogenetic Methods and Findings in Human Solid Tumors (by Floyd H. Thompson) you will find detailed protocolls for chromosome preparation (culturing, harvesting and staining) from solid Tumors!

One very importent comment on what Ajay was telling you: If you prepare your slides for chromosome preparation you should always prevent the cells from bursting if you drop them on the slide!!!! Why? Well, If they burst after hitting the slide all the chromosomes from that cell will be spreaded over the entire slide! In German this is called Chromosome-Salad and has nothing to do with the process of normal "chromosome spreading"! A mtotic cell doesn't have a nuclear envelope (membrane) anymore and therefore all her chromosomes are floating around the cytoplasma  motion-restricted by only one thing: the cell membrane! And if she gets lost the chromosomes will get lost too meaning swimming like a fish in the water and setteling somewhere on the slide! Take this always in mind while preparing chromosome slides!

Best regards Christian