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now: Wed Oct 18 21:38:54 2017 ... mod: Wed Dec 9 16:09:34 2015
Hmmmmm . do you think that at some point in your santimonious life that you, or at least one of your anrcetoss may have eaten an animal product (and here I cannot resist saying that I have never eaten a LIVE animal as you wrote in your post)? The plain fact is that modern man got here by eating meat and what other meager ingredients that he was able to raise for himself and his family. The rest of the world suffered from famine because modern farming methods that can produce surplus' crops to send to those starving countries did not come along until the industrial revolution when modern implements made it possible to clear large areas of land, till and cultivate the soil, and harvest the resulting crops I'm willing to bet, even now, that any one of the hungry of this world will not turn down a meal made up entirely of dead animals so get over yourself.While I'm talking about corporate farm methods, may I mention all of the untested chemicals that caused ill effects from paper-thin eagle's eggs (result of DDT contamination) to birth defects and cancers in humans that were rampantly used and are even now only being found as dangerous when we can get past the lobbiest's influence on our law makers and regulators such as the FDA if you want an example of gross malfeasance look at how aspartame was refused safe status for over 8 years and suddenly and suspiciously was granted approval by a new FDA head that summarily negated all objections and approved it's use, and then resigned and went to work for Searle the company that developed it at a very high salary and when the government finally appointed people to investigate the case, the two prosecutors found that there was insufficient evidence to come to an investigative conclusion. But then guess what? Those same two prosecutors resigned their government jobs. Their new employer Searle.However, that is sustenance with which to feed another blogpost. I have gotten off of the original reason I replied to your post, to wit: Get off of your self-elevated, golden soap box, get your nose out of the sky (it may rain and drowning is a distinct possibility with it poked that high up) and try to contain your supercilious air that is so common to those of your pompous ilk. I get tired of being low-rated because I enjoy a fine medium rare steak, grilled to perfection, over almost any other food out there. I also enjoy vegetables too nothing goes better with that steak than a good ol baked potato. If you choose the vegan life style, good, that is certainly your prerogative; but don't look down on others that don't subscribe to the same misguided logic that you do.
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
- 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)
- 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
- 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
- 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
- Wash 3x in 5ml of 3:1 methanol:acetic acid fix.
- Resuspend cells in fix to be slightly translucent (similar to scotch tape)
- 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
- After the droplets have run off of the slide immediately drop 2-3 drops of fix solution on the slide.
- Stand the slide up vertically and blot the edge to remove excess fix solution.
- Fan the slide once in the air and stand it vertically until it begins to dry.
- When the fix solution begins to evaporate place the slide on a 45C heat block.
- After a few minutes the dry slide can be stained.
Acid etch slides
- 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)
- submerge slides in 95% EtOH 3X
- wash slides 3X with H2O
- store slides in H2O at 4C
- leave slides at room temperature for 1-2 days
- stain with 1 μg / ml Hoechst 33258 for 5 minutes
- wash with 2x SSC (pH 7.8)
- mount a coverslip with 2x SSC (pH 7.8)
- heat on a hot plate 75C for 10 minutes
- treat with a 20W black-light at 75C
- wash with distilled water
- treat with 0.1 M Sorensen buffer with 6% Giemsa stain, 4 minutes
- heat slides overnight at 65C
- treat with PBS(-)1 37C, 2 minutes
- 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
- treat with 0.1 M Sorensen buffer with 6% Giemsa stain, 4 minutes
PBS(-) is phosphate buffered saline with no calcium or magnesium added
thymidine: 25 mg/ml
BrdU: 2 mg/ml
Colcemid: 10 μg/ml
Hoechst 33258: 0.25 mg/ml in H2
O Hoechst 33258 is insoluble in high concentration in phosphate buffers
Sorensen buffer, 0.1 M, pH 6.8: mix equal volumes 0.1 M Na2
and 0.1 M KH2
Thymidine block (optional)
Cells can be partially synchronized with thymidine to enrich for metaphase cells.
- add thymidine to 300 μg / ml for 15 hours
- the addition of thymidine arrests cells at the start of S-phase
- 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
- after the cells have been treated with fix for the first time, it can be helpful to do a few cycles of: spin-down, aspirate old fix, resuspend in new fix (by pipetting up and down). This can be done easily by transferring the cells to a microcentrifuge tube and spinning them down for 1 minute at 15000g (this doesn't damage them at all).
- fixed cells can be stored forever at -20C in the fixative. spin them down and and resuspend in fresh fix before use