We are assessing the genotoxicity of my nanoparticles using chromosomal aberration assay in Swiss albino mice. We have tried optimizing the procedure using i.p. administration of colchicine at dose of 4mg/kg and 3mg/kg 4 hour prior sacrificing the mouse.
1. The bone marrow cells are isolated by centrifugation (10,000rpm – 5mins) in FBS.
2. The isolated cells are treated using a 0.075M KCl solution for 30min at 37oC and fixed using acetic acid:methnol (1:3) 10 min at RT. The cells were then centrifuged at 2000rpm for 5mins and again resuspended in fixing solution
3. The smears were prepared by keeping the slide at 45o and the cell suspension was dropped from a height of 2-3cm followed by air dry and stained using Giemsa stain (5%w/v) for 30min.
4. The chromosome spread we obtain in not satisfactory for counting, as they are not distinct.
Any leads will be highly appreciated
Try placing smears in Thermotron (or analogous) at 23 degrees celsius and 46% humidity to aid spreading of metaphases. Also during fixation on the slide spread, exhaling on the slides to create moisture in between fixation steps also helps ( apply fixative once moisture evaporates). Leave slides to age for 1 week and the day before staining, leave overnight at 60 degrees celsius in a dry oven. Allow to return to room temp before staining. Hope it helps!
Thank you for your valuable response! What I understand here is that essentially I have to modify the smear preparation technique and the steps for fixing the cells is appropriate?
If you are not getting good metaphase spreads I would troubleshoot by changing the smear prep technique accordingly. Might be worth trying. Do anybody else agree?
Thank you, Dr Dario. I would give the above technique a try. In addition, if you have observed the attached image above there are very less cells in metaphase, is that because of inefficient hypotonic treatment?
Hi Chintan,
Hypotonic treatment will influence the spread of the metaphases, not the amount. I would improve hypotonic treatment because from the image you shown, chromosomes are in the cytoplasm. We typically blow air with a Pasteur pipette when adding the hypotonic solution to the cells and also during addition of each fixative step (be careful, blow very gently!).
Another improvement for a better spreading of chromosomes is the height of dropping the cell suspension. We drop from about 20-30 cm onto ice cold wet slides (slides are kept for at least 4 hours in the fridge in water before use).
Of note: I never used the technique for mice bone marrow cells, only for cultured human lymphocytes but the spreading technique should not differ much. Slide preparation is the most tricky step there.
Hi Christine,
Thank you for your valuable suggestions!
What I follow here is, after adding the hypotonic solution, I aspirate the cells with the micropipette. Is this not sufficient?
In addition, I read somewhere that the colchicine dose affects the chromosome spread and thus we decreased the dose from 4mg/kg to 3mg/kg and increased the time of hypotonic treatment from 30min to 1hr, however, we are still unable to count the aberrations
Further insights are appreciated
I think the problem with the dose may rather be the toxicity than a difference in spreading. It's always a bit tricky to get the right concentration, causing enough aberrations but not too cytotoxic.
Haven't you added negative controls to your experiment, how do they look like?
I am currently working with the negative controls itself. What we are trying to is to optimize the dose of colchicine and protocol for smear preparation. The attached image is of the negative control itself!
With 3mg/kg of dose and dropping from 20cm height helped us to resolve the spreads; however, not that much that it makes them easy to count. We are going to reduce the dose of colchicine down to 2mg/kg and observe the spread.
Let's hope that 2mg/kg works out!
Hi Chintan,
I do not see the image of your negative controls.
Just that you have an idea of what I mean, here a picture of one of our slides with objective 20x, the second with objective 100x (the one we use for evaluation), but, as already said, from cultured human lymphocytes (Whole blood)
The image attached here and the one attached with the question are from the negative control.
The one attached with this reply is prepared by dropping from the height of 20cm (1st image is at 40x objective and the 2nd one represents 100x objective). The chromosomes are condensed in each trial and we are unable to count. Another issue I see is the chromosomes are too small to be counted, isn't it?
The study is being conducted on Swiss albino mouse, if that helps.
Centrifugation in the first stage can be avoided.Simple aspiration in hypotonic with a syringe without needle will give a suspension.Keep in hypotonic for 30 min .Then separate cells by slowcentrifugation 800 rpm.discard supernatant and fix resuspended pellet by dropwise addition of cold fixative with shaking.keep in cold for 30 min with shaking.centrifuge and discard supernatant.fix the pellet again and repeat 4or5 times.Then make smears by dropping from 2 feet
Thank you for your kind inputs, Asha madam!
Avoiding the centrifugation step leads to less density of cells available. We have previously tried isolating the cells from the bone marrow through mere aspiration, however, the cell count is low. The rest of the techniques suggested by you is what we are using, however to no avail. We are still not able to get a countable chromosomal spread.
No don't avoid centrifugation but do at a slow speed 800rpm.We worked on effect of chloropyriphos on rat bone marrow chromosomes in a UGC minor project and got excellent spreads
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