help on G banding optimization

[JS]

Hi everyone,

I am doing G-banding on DLD cells using Gibco's 2.5% Trypsin.
I diluted my Trypsin in 0.15M NaCl to a working concentration of 0.025%.

The attached has been incubated in working conc. Trypsin for 3mins, and I'm getting either bandless or overtrypsinized chromosomes.
But I couldnt get any bands even if I alter Trypsin incubation time / Giemsa incubation time.




Could anyone provide some insights, please?


PS. The slides were preheated on hot plate at 65oC overnight.

[vantuinen]

We use Gibco 0.25% trypsin in Sorenson's (phosphate) buffer after baking slides for 1hr at 90 degrees.

[Giorgio Paskulin]

Both images seem undertrypsinized, even the overtrypsinized one.
Maybe you should adjust the pH of the NaCl/trypsin solution to 6.8.

Regards
Giorgio Paskulin


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[Dunn, Betty]

Hello,

 

There are a couple of issues that you might address to get better results.

 

The first image has lots of separated chromatids.  It’s been my experience that chromosomes with chromatids separated this much do not ever produce good bands, even if you can get any banding. 

 

The second image appears to me to have quite darkly stained chromosomes with smooth edges.  I would interpret this as under trypsinized not over trypsinized.   Over trypsinized metaphases appears variable depending on just how much over trypsinized the specimen.  Just a little bit over trypsinized one would expect to see bands, but the edges of the chromosome and the bands would appear rough – like the edges are eaten away.  Really, really over trypsinized chromosomes appear ghost-like – very pale in the middle of the chromosome/chromatid with darker chromosome and/or chromatid edges so an outline of the chromosome/chromatid is seen.   In between slightly over trypsinized and really over trypsinized is a gradual gradient between the two extremes.

 

Try for longer chromosomes without separated chromatids (lower colcemid concentration and/or exposure time).   This will produce fewer metaphases, but they should be much easier to work with.   Even with these short chromosomes without split chromatids try a stronger concentration of trypsin to get bands.      

 

I can’t tell from these stained images how flat the metaphases appear on the slide.  That must be assessed with phase optics.  If they appear cytoplasmic by phase (refractile) a greater trypsin exposure time is required to get any banding, but the banding will never be very good.   Good G-banding requires metaphases flattened during the drying step so there is approximately the same thickness of cytoplasm across the entire metaphase. 

 

Once you determine the Giemsa incubation time for this specimen type that exposure time will be very constant and will not need changes from day to day. 

 

The overnight aging temperature is one that is used frequently with good success so would not be an issue here.   

 

Good luck with improving your procedures.

 

Betty

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