Bone marrow culture-problem with elongation metaphase

[chinlee]

Hi,

I’m doing BM for overnight culture and synchronize culture (FDU/Uridine with Thymidine). We culture without 5% CO2. On average our bone marrow chromosomes are within 300-350 or most of the time even lower than 300. In this group discussion, many have mentioned usefulness of applying metaphase arresting solution (MAS) and chromosome resolution additive (CRA) in their culture. I want to try these reagents and hopefully can improve the resolution and mitotic index.

May I know is that necessary 5% CO2 incubation for overnight or synchronized culture?

Is that preferable using T-flask when culturing (5mL or 10ml media)?

 

For MAS: How many mililitres of MAS is needed in 10mL culture media and how long the incubation needed for synchronize culture or overnight culture?

 

For CRA, do you added pre or post colcemid (or MAS)? How long? How many mililiter (or follow as recommended by manufacturer 0.1mL for 10mL media)

 

Very much appreciate, could anyone please let me know the protocol step by step.

 

chinlee

[Dunn, Betty]

I'm addressing only the first two of your questions.

As long as your short term cultures (blood & bone marrow) are capped tightly (closed) they will survive and cells will divide for a few days.  The cells metabolism will produce sufficient CO2 to maintain the required media pH for cellular growth.  CO2 is needed from some outside source if cultures are not closed tightly or are long term cultures. 

The major advantage to using flask is that there are more cells placed in culture in a 10 ml volume so generally more cells are available for harvest and analysis.  Also, flask provide a greater surface area for media contact with air.  This contact area is where the buffer in the media and CO2 in the air interact to maintain a supportive pH for cellular division within the media.  Tubes can be incubated at a slant to increase the surface area contact to achieve the same result.   

Betty 

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From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] on behalf of chinlee [karun...@googlemail.com]
Sent: Monday, November 04, 2013 4:42 AM
To: cytogenetics-methods...@googlegroups.com
Subject: Bone marrow culture-problem with elongation metaphase

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[Phan Chin Lee]

Hi,
Betty,
Thank you very much.
This mean,  If 72 hours culture (in flask) without CO2 supply but capped tightly, the yield of cell and quality of metaphase will reduce or 3 days culture still acceptable without CO2 supply but no longer than 3 days culture.

chinlee

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From: DUN...@uthscsa.edu
To: cytogenetics-methods...@googlegroups.com
Subject: RE: Bone marrow culture-problem with elongation metaphase
Date: Wed, 6 Nov 2013 14:54:43 +0000

[Dunn, Betty]

Chinlee,

Your closed, short term cultures should survive with no reduction in quality or quantity of metaphases for 3 and maybe for 4 days, but no longer, as long as your media contains a sodium bicarbonate buffer (NaHCO3). 

You did not mention the concentration, amount added/5 ml culture media or the exposure time of your arresting agent.  All these can contribute to the short chromosomes you are getting, but BM chromosomes are usually shorter than from other tissues.  You might check your solutions against published protocols.  Do you have a copy of the AGT Cytogenetics Manual? 

Betty 

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From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] on behalf of Phan Chin Lee [cua...@hotmail.com]
Sent: Friday, November 08, 2013 2:24 AM

[Phan Chin Lee]

Thank you very much.
Take notes for other factor.
I don't have the AGT Cytogenetics Manual. In search through net, the 4th edition currently is in press. May I know do you have any information about he 4th edition  publication?

regards
chinlee

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From: DUN...@uthscsa.edu
To: cytogenetics-methods...@googlegroups.com
Subject: RE: Bone marrow culture-problem with elongation metaphase

Date: Fri, 8 Nov 2013 15:29:18 +0000

[Dunn, Betty]

Hi Chinlee,

I've heard nothing further about the expected publication date for the 4th edition.  It's certainly been a very long time coming.  Those at the annual meeting last June were assured it would be this fall.  We've now been waiting for years, so I do understand people's impatience.  I sure hope the wait will have be worth it!   

I still find the older editions do have very valuable information regarding the basics of cytogenetics.  It might be possible to find an older edition for sale at a good price on the web.  I've kept all the editions and sometimes still look through one of the oldest versions to find information lacking in one of the newer editions. 

Again, let's hope the 4th edition is out soon!

Betty   

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From: cytogenetics-methods...@googlegroups.com [cytogenetics-methods...@googlegroups.com] on behalf of Phan Chin Lee [cua...@hotmail.com]

Sent: Sunday, November 10, 2013 7:11 PM

[sindhu G]

Hi chinlee,

            5% CO2 is necessary for BM samples. can you please give me the information about metaphase arresting solution. If it is colchicine, may be it won't effect that much on your cells. my suggestion is to use colcemid to get better results.

           And my one more question is, How and Why you are suspecting reagents which you are using?

          I HOPE THIS WILL HELP YOU........

Sindhu

[Phan Chin Lee]

Hi Betty,
Yes, will try to get the 3rd edition while waiting for the fourth.
Thank you so much
chinlee
 

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From: DUN...@uthscsa.edu
To: cytogenetics-methods...@googlegroups.com
Subject: RE: Bone marrow culture-problem with elongation metaphase

Date: Tue, 12 Nov 2013 18:23:45 +0000

[Phan Chin Lee]

Hi Sidhu,
We use colcemid.  Try to improve chromosme quality. Tq
chinlee

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Date: Wed, 13 Nov 2013 02:55:08 -0800
From: nagasi...@gmail.com
To: cytogenetics-methods...@googlegroups.com
Subject: Re: Bone marrow culture-problem with elongation metaphase

[vu hanh]

Hi ChinLee
In bone marrow culture - problem enlongation metaphas
How do you use colcemid? I often add 50µl / 10minutes / 10ml culture media (concentration 10µg/ml Gibco).
I often culture bone marrow without CO2 5%/ 24h and we still have a high mitosis rate. If you culture > 72h CO2 5% is neccesary but in bone marrow even we can culture in 4h.

[Phan Chin Lee]

Hi,
we set-up two cultures
1.overnight culture with colcemid (ONC) :25uL colcemid per 5mL culture and incubate overnight (colcemid 10µg/ml Gibco)
2. the other is synchronized culture: 50uL colcemid per 5mL for 30minutes.
without CO2 5%.
Probably we will perform 24hrs culture to replace ONC. We want to improve metaphase quality and chromosome morphology. yes, will look into CO2 5% too for culture more than 3-4 days.
regards
chinlee

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Date: Wed, 11 Dec 2013 04:38:23 -0800
From: vuhan...@gmail.com
To: cytogenetics-methods...@googlegroups.com
CC: cua...@hotmail.com

Subject: Re: Bone marrow culture-problem with elongation metaphase

[Vũ hạnh]

Hi!

Oh,Both of the two ways you're using not well. You should change the way use colcemid to purpose with your lab condition.
I test many ways using colcemid and see that use colcemid before haverst is still best.
But I think you need to check culture and haverst steps.

How do you culture and haverst? for example: cells / 1ml media (very important)...
My lab culture for CML, AML , ALL are good with high abnormal chromosome rate but myeloma multiple detect abnormal chromose is too low and we use IL-4 , IL-6 in culture, now

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[Phan Chin Lee]

The yield is alright, if possible we want to get longer chromosome. We are improving now, base on all information given by members of the group.
Thank you for all the input and suggestion.
regards,
chinlee

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Date: Fri, 13 Dec 2013 23:44:07 +0700

Subject: Re: Bone marrow culture-problem with elongation metaphase

From: vuhan...@gmail.com
To: cytogenetics-methods...@googlegroups.com