Hello Abdelkareem Ahmed

The cells need to be delicate so that they will burst when dropped onto a microscope slide. To achieve this, you need to add a hypotonic solution to the cells, which has a lower concentration of solutes relative to the inside of the cell. This causes an osmotic gradient to form between the outside and the inside of the cells. Thus, adding the hypotonic solution causes the cells to swell, making them very fragile.

The easiest way to achieve this balance is to add the hypotonic solution a little at a time, rather than the whole amount at once. In this way, you can resuspend your cells slowly, allowing for uniform swelling. You may want to make sure that there are no clumps of cells in your tube. Gently tapping on the tube should break up any unwanted cell clumps. The most common hypotonic solution for metaphase spreads is potassium chloride, but some protocols use sodium citrate.

You should think of the cells like water balloons. You may want them full enough so that they’ll burst when they hit something, but be a little careful, not so full enough to break in your hands!

You may want to refer to the articles attached below. These may be helpful.

https://www.jstage.jst.go.jp/article/csf1975/1/1/1_1_93/_article

Article A reliable protocol for high-quality metaphase spreads for i...

Best.

@ Malcolm Nobre

Hello mr. Malcolm

We do use 0.075M potassium chloride solution as a hypotonic solution.

We flush out the bone marrow from femur of the mouse with an isotonic solution and centrifuge the tubes at 10,000 rpm for 10 minutes.

Then we add hypotonic drop by drop to the tube containing the pelleted cells and incubate them for 30 minutes at 37°C.

After that we centrifuge again at 10,000 rpm for 10 minutes and add 5 ml of methanol:acetic acid fixative drop by drop and repeat centrifugation and fixation two more times.

Please note that when adding hypotonic or fixative drop by drop we vortex the tubes during the whole process.

Could the rpm speed of centrifugation be harsh for the cells that it shrunkens the cells again?

Or could it be the vortexing of the tubes? Or both?

Note #2: we are doing normal light microscopy with no fluorescent dyes or special types of microscopy. We use just giemsa staining.

Thanks in advance.

Here are pictures of the cells under microscope.

These slides were prepared according to the previously mentioned protocol and yet they look too small and don't burst.