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ChromosomeSpread
ChromosomeSpread

Chromosome Spreads*

Cells are first synchronized in S-phase by thymidine block prior to metaphase arrest to enhance the percentage of cells in metaphase. Chromosomes are then spread and fixed on slides and treated with various band-staining procedures.
*basic protocol courtesy of Xiao (Tracy) Cui

Arresting cells in metaphase

  1. add Colcemid to 0.02 μg / ml for 3 to 4 hours (or longer -- see below)
    • the longer the Colcemid is added, the higher the percentage of cells in metaphase will be, however, if cells are arrested in metaphase by the Colcemid for long periods of time, the chromosomes will condense and shrink somewhat, which can make assays such as FluorescencePlusGiemsa staining for sister chromatid exchanges more difficult to score. Even so, a longer time in Colcemid for slower growing lines may be warranted
    • optional: synchronize cells with thymidine prior to colemid treatment (see below)

Harvesting metaphases

  1. remove and save the culture medium, treat cells with 0.05% trypsin until the majority of the cells 'round-up', add back the medium to stop the trypsin.
    • The trypsin reaction can be slowed down by dilution in PBS by 1:5 or 1:10. This can be helpful in making sure that a lot of non-metaphase cells do not get detached from the plate
    • adherent cells in metaphase 'round-up' and are more easily removed from the plate than cells not in metaphase. by light trypsin treatment the goal is to free the metaphase cells while leaving the non-metaphase cells attached to the plate. look for only a small amount of cells to be released and the majority still attached. it may also be possible to carefully monitor the rounding-up procedure and "blow off" the rounded metaphase cells by vigorous pipetting
    • the culture medium must contain serum to inhibit trypsin
  2. Spin cells down and resuspend them in 10 mL of hypotonic solution: 46.5 mM KCl / 8.5 mM Na⋅Citrate, 37C, 20-25 minutes (this time is also approximate -- see below)
    • the goal is to make the cells swell up. cells will appear spherical under the microscope when sufficiently swollen. proceed directly to the next step when that happens as too long in hypotonic solution can lyse delicate cells
  3. add 1 mL freshly made fix (3:1 methanol:acetic acid) and mix well. Spin down, resuspend in 5 mL fix, let stand for at least 15 minutes
  4. Wash 3x in 5ml of 3:1 methanol:acetic acid fix.
  5. Resuspend cells in fix to be slightly translucent (similar to scotch tape)
  6. Using a pasture pipette quickly drop 4-5 drops of the cell-fix suspension onto a still wet acid etched slide(read below) that has been stored at 4C in H2O. The slide should be placed on a slope such that the droplets run off of the slide length-wise. The correct angle for this can easily be accomplished by placing one end of the slide on a 10 mL disposable pipette.
    • store slides submerged in H20 at 4C
    • the cells break open when the fix solution evaporates, not from the force of a dropping impact. The longer it takes for the fix to evaporate, the more spread the chromosomes will be. Increase the time for evaporation by (for example) keeping the slide in a humid environment (such as over a waterbath), and/or by pre-chilling the slide. It is possible to spread the chromosomes too much such that they no longer group as discrete metaphases
  7. After the droplets have run off of the slide immediately drop 2-3 drops of fix solution on the slide.
  8. Stand the slide up vertically and blot the edge to remove excess fix solution.
  9. Fan the slide once in the air and stand it vertically until it begins to dry.
  10. When the fix solution begins to evaporate place the slide on a 45C heat block.
  11. After a few minutes the dry slide can be stained.

Acid etch slides

  1. submerge slides in etching solutions for 15-20 minutes. Etching solution: 0.1N HCL in 95% EtOH (720 mL EtOH + 80 mL 1 N HCl)
  2. submerge slides in 95% EtOH 3X
  3. wash slides 3X with H2O
  4. store slides in H2O at 4C

R-banding

  1. leave slides at room temperature for 1-2 days
  2. stain with 1 μg / ml Hoechst 33258 for 5 minutes
  3. wash with 2x SSC (pH 7.8)
  4. mount a coverslip with 2x SSC (pH 7.8)
  5. heat on a hot plate 75C for 10 minutes
  6. treat with a 20W black-light at 75C
  7. wash with distilled water
  8. treat with 0.1 M Sorensen buffer with 6% Giemsa stain, 4 minutes

G-banding

  1. heat slides overnight at 65C
  2. treat with PBS(-)1 37C, 2 minutes
  3. treat with 0.025% trypsin in PBS(-), 37C, 60 seconds
    • if no bands are seen, trypsin treatment needs to be increased
    • if chromosomes are swollen and indistinct, trypsin treatment need to be decreased
  4. treat with 0.1 M Sorensen buffer with 6% Giemsa stain, 4 minutes
1PBS(-) is phosphate buffered saline with no calcium or magnesium added


Reagents

thymidine: 25 mg/ml
BrdU: 2 mg/ml
Colcemid: 10 μg/ml
Hoechst 33258: 0.25 mg/ml in H2O Hoechst 33258 is insoluble in high concentration in phosphate buffers
Sorensen buffer, 0.1 M, pH 6.8: mix equal volumes 0.1 M Na2HPO4 and 0.1 M KH2PO4


Thymidine block (optional)

Cells can be partially synchronized with thymidine to enrich for metaphase cells.
  1. add thymidine to 300 μg / ml for 15 hours
    • the addition of thymidine arrests cells at the start of S-phase
  2. wash cells 2x with PBS, then 1x with pre-warmed culture medium, then aspirate and add back complete, pre-warmed medium without thymidine for 5-6 hours.
    • the 5-6 hour incubation is to allow cells to unarrest from the thymidine and transit to M-phase. this time may be longer for slower growing lines
    • if R-banding: supplement the medium with BrdU (bromo-deoxyuridine) to 10 μg / ml
    • it may be useful to directly add the Colcemid from the next step (see below) to the medium at this time. Colcemid will not affect cells in S-phase. for adherent cells, monitoring cells under the microscope for the "rounding up" characteristic of M-phase may be useful in timing this step


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