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.
 
Outline protocol for staining nuclei for DNA measurement using flow cytometry

Materials and Reagents

Double-edged razor blades (much thinner and sharper than the single-edge type usually found in laboratories).

Chopping buffer:
45 mM MgCl2 (MW of hexahydrate: 203.3)
30 mM Na3 citrate
20 mM MOPS (3-[N-mopholion]propanesulphonic acid)
0.1 ul/ml Triton X-100.

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
irregular chopping make the peaks from cytometry less sharp. Suck the chopping buffer and freed nuclei into a syringe through
the nylon Miracloth, and squeeze into the analysis tube through another piece of cloth. Put onto flow cytometer!

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}
take write-able CD!

USEFUL WEBSITES
The site of Jaroslav Dolezel has extensive information about flow cytometry and its application to plants: http://www.ueb.cas.cz/olomouc1

The site http://flowcyt.cyto.purdue.edu/flowcyt/software.htm has a listing of free cytometry software.

Our publications on aspects of flow cytometry: Link to our full publication list

134. Schwarzacher T, Wang ML, Leitch AR, Miller N, Moore G, Heslop-Harrison JS. 1997. Flow cytometric analysis of the
chromosomes and stability of a wheat cell culture line. Theor.Appl.Genet. 94: 91-97.

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
fifteen clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year while a parallel
subculture had a slightly different, stable, karyotype following different growth conditions. The data show that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow histogram peaks, so the method is useful for flow sorting and the construction of chromosome-specific recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes.

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
reviews genome size analysis - changes between species, during differentiation and during the cell cycle, and chromosome sorting for gene mapping and library construction.

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
are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A.
The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.

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
than an unreplicated (1C) bread wheat genome. In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as
tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TaKB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. The in situ hybridization pattern and chromosome morphology allowed 27 chromosome
types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. An in situ analysis of rDNA in the
TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.

Please send corrections, comments or questions PHH4@le.ac.uk