Why is fetal calf serum necessary for this assay? When performed from bone marrow why do the cells need to be re-suspended in FCS? The methanol fixation is done after smears are made...so I don't see the point of using FCS!
According to me the only thing you can do to perform this assay is to prepare a blood smear, to fix it with methanol or ethanol (depends on species used) and then stain with Gimmsa. You don't need to use FCS!
Well i could use plane PBS? But what volume of medium should I use? 0.5/1 ml per bone?
Especially for the micronucleus assay with rat bone marrow erythrocytes we replace FCS for suspension of the bone marrow cells by a special medium to avoid artefacts due to for example mast cell granules which can stain like micronuclei on the slides. We therefore use HBSS + 1.0% (W/v) bovine serum albumin + 0.15% (w/v) EDTA, pH7.4. This medium performs well in our hands. The medium was published by Agarawal & Chauhan (1993), BIOTECHNIC & HISTOCHEMISTRY, Volume 68 (4):187-188
Dear dr Ziemann, thank you for useful tips (both medium and reference to cite) I'm very grateful
Dear Nikola,
The use of Fetal calf serum in micronucleus test is to digest some connective tissues that may prevent observation of the cells in their original shape and form so as to see the nuclear content very well. This will reveal any micronuclei that may be present in the cells after staining with the appropriate dye. However, apart from FCS, PBS and other solutions previous researchers might have said, sodium citrate solution (2.2%) can also be used.
thanks.
Thanks everyone. FCS is traditionally used because it does not contain many electrolytes so doesn't become too hypertonic on drying. Other forms of serum should work just as well but might be improved by heat inactivation (56°C for 30 mins). Hanks' BSS can be used but expect some of the cells to lyse. Bovine serum can be substituted as a protectant.
When using rat bone marrow (as opposed to mouse), it helps remove some of the extra-cellular debris. It will not remove all the mast cell granules found in rat bm. Therefore, acridine orange fluorescent staining is used for rat as opposed to Giemsa. AO also gives good differentiation with blood; a supravital staining method can al;so be used with blood. My 1996 paper Evaluation of the rat bone marrow and peripheral blood micronucleus test using monocrotaline gives detailed methods. The Feulgen method is fussy so I would stick to AO or Giemsa.
Dear Raymond thank you for your answer.
Im using mice for this test and i have tried to do it with HBSS-BSA and with citrate solution. The HBSS-BSA worked fine (in control groups) no cell were attached to each other on the opposite all that happened in citrate solution.
I have stained the slides with both Giemsa and Feulgen method and I just glanced on those slides...didnt have time to devout to detailed analysis jet! But thank you for the guides and I will read the paper before further experiments most certainly. Also fluorescent dying is to expensive for our conditions for the moment.
Please see the attached file as an additional information
Article Antigenotoxic Potential of Rutin and Quercetin in Swiss Mice...
The protein in the FCS is protective. As the cells dry out in the smear any dissolved material becomes more concentrated. The dissolved salts in particular create a hypertonic medium which withdraws water from the unfixed cells and distorts them. Proteins (as in serum) protect the cell membrane and tend to create a low hypertonic effect on the cells. So FCS isn't needed to wash the cells but it is useful for making the final cell suspension prior to smearing. In the rat micronucleus test, the bone marrow has lots of extra-cellular debris so an additional wash in Hanks' balanced salts solution or similar is necessary to clean it up as per Mutat Res. 1997 Aug 14;392(3):243-9. Evaluation of the rat bone marrow and peripheral blood micronucleus test using monocrotaline. Proudlock et al.
In practice, the final suspension can be made in Hanks' or similar but the erythrocyte morphology isn't as well preserved. Other types of sera or protein solutions (e.g. bovine serum albumen or even plant albumen) probably give just as good results if used at the right concentration. The presence of protein in the final smear may prevent cells from floating off the slide during staining with Giemsa or supra-vital with AO.