Harvesting long chromes

[perla natalia Vargas]

Dear community, 

I am new and so glad to be here!

I have a question for you...

I am trying to sync my cell cycle in order to achieve high resolution chromes by starting my harvest when chromes are in prometaphase. 

How many hours should my chromes be in culture in order to achieve this? 

Also, I am shortening my colcemid exposure to 20mins (conc being 50ul)

No THY or MTX used. 

Looking forward to your comments!

[Kath]

Hi Peria,

I am new here to forum also. Saw your post. We have been adding  a elongation additive called CRA reagent, purchased from Rainbow Scientific, for several years now on all our cancer cytogenetics  tumor cultures in order to improve the chromosome resolution.  Its worth investigating. Try reducing your colcemid time further to 10 -15 minutes, this might also help you. You didnt mention what specimen type you are using? Let us know how you go.

Kath

[Paul Oei]

Try using echinomycin.  Look up the references, resolution is good and provides good banding under normal GTG compared to other techniques.  

Cheers

Paul

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[perla natalia Vargas]

Dear all, thank you so much!

I will look into both options then. 

Kath, you recommend reducing my colcemid time but keep my conc to 50ul ? (sample vol: 250ul, 5ml media)

Thank you so much!!

[perla natalia Vargas]

This is totally unrelated but so important as well...

I can't seem to get good looking chromes no matter what I do: cytoplasm is there and they look black under phase scope. 

Once I add trypsin they are usually overtrypsinized. 

my thermotron setting is as follows: 25C and 53% 

my trypsin conc: 0.25% 

trypsin time is usually 1;10-1:30min 

trypsin vol: 4ml in 46ml of hanks solution.

I don't know where my problem is: whether in dropping the slide or in banding.

Results are always dark lookin chromes that are overtryp, not under.

Looking for advice!!

Thank you so much!!!

PS: working on bloods. 

On Friday, March 17, 2023 at 4:38:33 AM UTC-4 Paul Oei wrote:

[DANIEL ANDRES FABIAN VILLAGOMEZ ZAVALA]

Hi Perla,

My advice to you is as follows: Thermotron settings should be 25C and 62 humidity. Trypsinization time 45 sec. For harvesting blood use 18 min of colcemid treatment and 20 min hypotonic treatment. Drop cells after 24 hrs of  harvesting them, they will be quite good fixed. 

hope this helps,

Daniel A. F. Villagomez

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[perla natalia Vargas]

Thank you so much!

Will le y your know how it goes!

To view this discussion on the web visit https://groups.google.com/d/msgid/cytogenetics-methods-and-trouble-shooting/CADGgned4B%3Dhi9wNXF4O2yk0fKF4pZ52UmR9QgYwkXQTQURvQxA%40mail.gmail.com.

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P. Natalia Vargas Schwartz, CG(ASCP)
Citogenetista y Bióloga Molecular 

Directora de ORIGEN - Laboratorio Genetico  
(+591)-708-11-444