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Flow Cytometry |
We are currently using the instrument for comparative measurement of DNA contents, ploidy and DNA amount analysis in oil palm and other species. Materials and Reagents Double-edged razor blades (much thinner and sharper than the single-edge type usually found in laboratories). Chopping buffer: To 20 ml, add 100 ul 1 mg/ml propidium iodide – making up enough for one days chopping and analysis, but not storing longer. Optionally, add RNase: DNase-free ribonuclease A (e.g. Sigma R4642, solution in 10 mM Tris-HCl, pH 8 and 50% glycerol, 70 units/mg protein). Make up stock of 10 mg/ml in 10 mM Tris-HCl, pH 8. Store at –20 C in aliquots. Dilute to 100 ug/ml prior to use. The RNase must be DNase-free. If RNase is not purchased DNase-free, inactivate DNase in the RNase by placing the stock solution in boiling water for 15 min. If plant material browns rapidly during chopping, you can add DTT (2.5 ul of 100 mM per 10 ml buffer) Miracloth or equivalent with a mesh size of 50-80 um. Method Chop fresh tissue in 1-2 ml of chopping buffer. About 5 minutes chopping of a small piece of leaf is sufficient - but slicing and General notes to analysis: We analyze the DNA content peaks using the free programme WinMDI (available from www.salk.org; http://flowcyt.cyto.purdue.edu/flowcyt/software.htm has links to an excellent selection of flow cytometry analysis software. Our CV for beads is about 1.7 to 2%. {Internal information University of Leicester: Malcolm Rae mjlr1 for bookings, £15/hr internal charge} USEFUL WEBSITES The site http://flowcyt.cyto.purdue.edu/flowcyt/software.htm has a listing of free cytometry software. 134. Schwarzacher T, Wang ML, Leitch AR, Miller N, Moore G, Heslop-Harrison JS. 1997. Flow cytometric analysis of the A rapidly growing, long term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine and a chromosome suspension with 2 to 3ÿxÿ106 chromosomes ml-1 was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin A3, univariate and bivariate flow cytometry histograms showed 119. Heslop-Harrison JS. 1995. Flow cytometry and genome analysis. Probe 5(1): 14-17. Flow cytometry allows for fast and informative, quantitative and qualitative analysis of objects including chromosomes and nuclei, normally by measuring the fluorescence of molecules that are specifically bound to structures of interest. The molecules measured are often fluorochrome-conjugated antibodies or fluorescent dyes binding specifically to DNA proteins. The article 99. Heslop-Harrison JS, Schwarzacher T. 1996. Flow cytometry and chromosome sorting. In: Fukui K, Nakayama S, eds. Plant Chromosomes: Laboratory Methods. Boca Raton: CRC Press. pp. 85-108. 62. Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G. 1992. Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes. Nucl.Acids Res. 20: 1897-1901. We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half the cloned DNA sequences analysed 61. Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, Surlan-Momirovic G, Moore G, Heslop-Harrison JS. 1993. Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture. Plant Cell,Tissue and Organ Culture 33: 287-296. A rapidly growing Triticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower Please send corrections, comments or questions PHH4@le.ac.uk
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