\n\t1. 100 µl EDTA coated blood collection tubes without capillary (Scientific Laboratory Supplies catalogue number 078042)
\n\n\t2. Centrifuge (for plates)
\n\n\t3. Dispensing troughs
\n\n\t4. Sterilin 96 V bottom plates with lid
\n\n\t5. Sterilin 96 flat bottom plates with lid
\n\n\t6. FACS buffer (PBS with 0.5% BSA)
\n\n\t7. Pipettes (single, multi and repeat) and filter tips
\n\n\t8. DNA dye waste container, plus funnel
\n\n\t9. Timer
\n\n\t10. 4 ml amber glass vials (Sigma catalogue number 27001-U)
\n\n\t11. BD LSRII flow cytometer
\n\n\t12. APC-Terr 119, PE-CD71 antibodies and Hoechst dye
\n","Procedure":"\n\tSample collection and preparation:
\n\n\ta. 49 µl of FACS buffer is pipetted into the required wells of a 96 well V bottom plate
\n\n\tb. 1 µl of whole blood from a 100 µl EDTA coated tube (collected by puncture of the retro-orbital sinus) is pipetted onto the plate. This is repeated for all samples.
\n\n\tc. 50 µl of 2x CD71/Ter119/Hoechst cocktail is added and incubated for 20 minutes.
\n\n\td. Cells are washed with FACS buffer before 25µl of the stained sample is transferred to a flat bottom plate containing 275 µl of FACS buffer. The plate can then be run on the BD LSRII flow cytometer.
\n\n\t
\n\n\t
\n\n\tAnalysis
\n\n\tData is analysed using FlowJo software. PE-A CD71 is plotted against Hoechst-A to distinguish and gate reticulocyte (RET), micronucleated reticulocyte (MN-RET), normochromic erythrocyte (NCE) and micronucleated normochromic erythrocyte (MN-NCE) populations.
\n","Purpose":"\n\tThe micronuclei assay is a mutagenic test that can identify increases in the frequency of normochromatic micronucleated erythrocytes, reticulocytes and micronucleated reticulocytes, all of which can act as markers for chromosomal damage. The assay can detect genotoxic events that occur during interphase, which may be the result of a disruption to the number of chromosomes (aneugenic), or by damage to the chromosomes themselves (clastogenic).
\n","Experimental Design":"\n\tMinimum number of mutant animals: 7 mice for each sex.
\n\n\tAge of animals: 16 weeks (fixed).
\n\n\tSexual dimorphism: yes for some of the parameters.
\n","Notes":"\n\tThe micronucleus assay is performed at 16 weeks of age
\n\n\tNon-fasted mice are terminally anaesthetised and blood is collected into EDTA coated tubes via the retro-orbital sinus. Whole blood is stained with a titrated cocktail of antibodies.
\n\n\tIn order to eliminate aggregated antibodies from the mix, briefly centrifuge the antibodies before adding them to the cocktail.
\n\n\t
\n"}