Dear fellow researchers,
While performing an in-vitro micronucleus assay with caco-2 cells I constantly obtain results with a very poorly defined and stained cytoplasma, which makes the evaluation of the assay very hard/impossible. Does anyone have similar problems or experience in dealing with this problem?
- During the assay Caco-2 cells are kept in DMEM-F12 containing 1, or 10 % FBS
- Cytochalasin B is added in a concentration of 4.5 microgram/mL
- hypotonic shock is perfromed with 0.075 M KCl drop-by-drop while constantly vortexing
- Fixation in 3:1 methanol: acetic acid
- The staining is with 4% Giemsa diluted in Gurr´s buffer
Thank you all very much.
Best reagrds,
Nicolaj
For how long did you leave the hypotonic solution to stand for?
What is the reasons for vortexing?
These are the major factors responsible for lost of cytoplasm in the cells during preparation.
I suggest you avoid adding 0.075 M KCl or add if necessary for a maximum of 3 min and neutralize the hypotonic effects immediately by adding fixative.
Do not vortex but gently contrifuge normally may be at 300 g per 5 min.
Consider increasing the concentration of Giemsa to 8-10%.
I hope you will find this useful.
All the best
Dear Mr. Alimba. Thank you for your response. The vortexing was added to guarantee a homogenous solution. The hypotonic solution was left on the cells for approx. 30 seconds (including the addition to the cell suspension. Before the whole cell suspension is centrifuged for 5 min at 1000 rpm
I will then suggest you avoid vortexing and make sure you add fixative to stop the action of KCl from hypotonizing the cells before you centrifuge.
Since you did not stop the action of KCl, it will still be active while centrifuging for 5 mins.
I suggest you try correcting this effects and moreso increase the conc of your stain to 8%.
i guess you will have a better result.
Dear Mr. Alimba, thanks for this advices. I'll try it and report if it improved the results.
Dear colleagues,
Here is a short update on this topic.
An increase of the concentration of hypotonic solution to 0.1M KCl (at 37 °C) and reducing the incubation time with it to approx. 30 sec., before adding the ice-cold fixing solution, improved the results.
An increase in Giemsa concentration did not change the display of the cytoplasm.
Most of the cells show a visible cytoplasm now and it is possible to score a majority of the cells, nevertheless further improvements in the method should be tested.
Best regards
It is good to know that it worked. However i will still recommend that you retain the concentration of KCl at 0.075 M (at 37 C) in case of not meeting up with 30 sec.
Furthermore, Giemsa was not meant to stain the cytoplasm but the Nucleus. The aim for increasing the Giemsa to 8% is to make the nucleus more conspicuously distinguished from the cytoplasm.
I wish you all the best.
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