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Flow Karyotyping and Chromosome Sorting

Univariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes stained with 4-,6-diamidino-2-phenylindole (DAPI). This method was originally developed by Lucretti et al. (1993) for field bean and requires a flow cytometer equipped with a light source providing excitation in UV. We use BD FACSVantage equipped with a Coherent Innova 305 argon-ion laser.

Flow Karyotyping

  1. Stain a chromosome suspension (approx. 1ml) by adding DAPI stock solution to final concentration of 2 µg/ml.
  2. Filter the suspension through a 20-µm nylon mesh.
  3. Make sure that the flow cytometer is properly aligned for univariate analysis. Make sure that the band-pass filter 424/44 is placed in front of the fluorescence 1 (FL1) detector.
  4. Run a dummy sample (LB01 buffer containing 2 µg/ml DAPI) to equilibrate the sample line.
  5. Introduce the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis!
  6. Set a gating region on a dot plot of forward scatter height (FSC-H) and FL1 pulse height (FL1-H) to exclude debris, nuclei and large clumps.
  7. Adjust photomultiplier voltage and amplification gains so that chromosome peaks are evenly distributed on a histogram of FL1 pulse area (FL1-A).
  8. Analyse 20 - 50 thousand chromosomes and save the result on computer disc.

Chromosome Sorting

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a dot plot of FL1 pulse width (FL1-W) versus FL1-A.
  3. Adjust the FL1-W amplifier gain and width offset as needed to achieve optimal resolution of the width signal.
  4. Check for stability of the break-off point and of the side streams.
  5. Define sorting region for the largest chromosome on the FL1-W versus FL1-A dot-plot.
  6. Select 1 drop sort envelope (number of deflected drops) and - counter - sort mode giving the highest purity and count precision.
  7. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  8. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
  9. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  10. Define a sorting region for the chromosome to be sorted on the dot-plot of FL1-W versus FL1-A.
  11. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted, and desired volume for the sorted fraction.
  12. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube).
  13. Briefly spin the tube at room temperature.
  14. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)

Bivariate Flow Karyotyping and Chromosome Sorting

This protocol describes the analysis and sorting of plant chromosomes after dual staining with 4’,6-diamidino-2-phenylindole (DAPI) that binds preferentially to AT-rich regions of DNA, and with mithramycin that binds preferentially to GC-rich regions of DNA. This method was originally developed by Lucretti and Dolezel (1997) for broad bean and requires a flow cytometer equipped with two lasers (one of them UV). We use BD FACSVantage equipped with Coherent Innova 305 and Coherent Innova 70C argon-ion lasers.

Bivariate Flow Karyotyping

  1. Add MgSO4   stock solution to chromosome suspension (approx. 1ml) to a final concentration of 10 mM.
  2. Stain the chromosomes in suspension by adding DAPI stock solution to a final concentration of 1.5 µg/ml and mithramycin stock solution to a final concentration of 20 µg/ml. Leave to equilibrate for 30 min on ice.
  3. Make sure that the flow cytometer is properly aligned for bivariate analysis. Use a half mirror to split the fluorescence of DAPI to FL1 detector through the 424/44 band-pass filter and mithramycin fluorescence to fluorescence 4 (FL4) detector through a 575/26 band-pass filter.
  4. Run a dummy sample (LB01 buffer containing 1.5 µg/ml DAPI and 20 µg/ml mithramycin) to equilibrate the sample line.
  5. Filter the sample through a 20-µm nylon mesh.
  6. Run the sample; let it stabilise at appropriate flow rate (e.g., 200 particles/s). If possible, do not change the flow rate during the analysis.
  7. Set a gating region on a dot plot of FSC-H versus FL1-H.
  8. Adjust photomultiplier voltages and amplification gains so that chromosome peaks are evenly distributed on histograms of FL1-A and FL4 pulse area (FL4-A).
  9. Display the data on a dot-plot of FL1-A versus FL4-A.
  10. Analyse 20 - 50 thousand chromosomes and save the result on a disc.

Chromosome Sorting

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a dot plot of FL1-A versus FL4-A.
  3. Check for stability of the break-off point and of the side streams.
  4. Define sorting region for the largest chromosome on the FL1-A versus FL4-A dot-plot.
  5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
  6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  7. Check the number of chromosomes using a fluorescence microscope (do not cover the drop with a coverslip!).
  8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  9. Define a sorting region for the chromosome to be sorted on a dot plot of FL1-A versus FL4-A.
  10. Select sort mode and sort envelope according to required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.
  11. Sort the required number of chromosomes into a polystyrene tube containing the appropriate amount of the collection liquid. The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use. For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube.).
  12. Briefly spin the tube at room temperature.
  13. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

References

Lucretti S, Dolezel J. Bivariate flow karyotyping in broad bean (Vicia faba). Cytometry 28: 236 - 242 (1997)

Two-Step Sorting

This protocol is used to sort chromosomes when their frequency in the original suspension is too low. This is frequently the case for large chromosomes, which break more easily than smaller chromosomes during chromosome isolation (Lucretti et al. 1993). During the first sort, the sample is enriched for the required chromosome. During the second sort, the chromosomes are sorted with a high purity. We use this protocol with BD FACSVantage dual laser flow cytometer and sorter.

First Sort

  1. Make sure that the sorting device is properly adjusted.
  2. Run the sample and display the signals on a suitable distribution.
  3. Check for stability of the break-off point and of the side streams.
  4. Define sorting region for the largest chromosome on a suitable distribution.
  5. Select 1 drop sort envelope (number of deflected drops) and counter sort mode giving the highest purity and count precision.
  6. Sort exact number of chromosomes (e.g., 50) on a microscope slide.
  7. Check the number of chromosomes using a fluorescence microscope (do not cover with a coverslip!).
  8. If the number is not correct, repeat adjustment of the sorting device using fluorescent beads.
  9. Select the enrich mode and 3 deflected drops sort envelope that allows for the highest recovery.
  10. On a suitable distribution, define a sorting region for the chromosome(s) to be sorted.
  11. Sort approximately 105 chromosomes into 400 µl of LB01T into a 1.5 ml sample polystyrene cup (Deltalab).

Second Sort

  1. Add fluorescent dye(s) to pre-sorted chromosome suspension to reach recommended final concentration(s).
  2. Run the sample and define sorting region for the chromosome to be sorted.
  3. Select sort mode and sort envelope. (Sort mode and sort envelope are selected according to the required purity, number of chromosomes to be sorted and desired volume for the sorted fraction.).
  4. Sort the required number of chromosomes into a polystyrene tube containing corresponding amount of the collection liquid. (The amount and composition of the collection liquid depends on the number of sorted chromosomes and on their subsequent use). For PCR, use a small amount (20 - 60 µl) of sterile deionized H2O in 0.5-ml PCR tube).
  5. Briefly spin the tube at room temperature
  6. Sort chromosomes onto a microscope slide for estimation of purity using PRINS.

Notes  
The actual number of chromosomes that should be sorted depends on the number of chromosomes that will be sorted during the second sort. It is recommended to sort at least five times more chromosomes than the final number required.  
In some cases, it may be practical to enrich the sample for more than one chromosome. Individual chromosomes are sorted during the second sort.

References

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. Theoretical and Applied Genetetics 85: 665 - 672 (1993)