Elongating Chromosomes for GTG-banding without Toxic Chemicals

[Valerie Tio (2402119161)]

Hi everyone,

I'm looking for advice on how to achieve longer chromosomes for GTG-banding without using toxic chemicals like ethidium bromide or actinomycin D. I've heard about a product called Chromosome Resolution Additive (CRA) by Rainbow Scientific that claims to be non-toxic and effective in improving chromosome spreading.

Has anyone used this product or have experience with other non-toxic methods for chromosome elongation? Any tips or recommendations would be greatly appreciated!

Thanks in advance,

Valerie 

[maria mouriki]

Hello Valerie. I have no experience of using this product but talk me through your issue, i may be able to help you. What kind of samples are you taking about? Lymphocytes, amniotic fluid, cvs, bone marrow? What protocol are you using at the moment? Give us some details. And provide us with some photos of unbanded metaphases and some banded ones.
Maria Mouriki

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[Valerie Tio (2402119161)]

Hi Maria. So glad to have you here as I saw that this group is no longer active! My lab is currently developing culturing protocol of amniotic fluid for GTG-banding. We use the   AmnioMax Basal and supplement medium from Gibco with 6:1 ratio (12 mL of basal + 2 mL of supplement). Currently, I do not have any picture of the unbanded metaphase. I will attach the ones for banded metaphase. We have few problems from amniotic fluid, like inconsistent harvest results. Sometimes we get a lot of metaphase and long one, sometimes none at all, low mitotic index and split sister chromatids, but we always did the same protocol. The thing is, we encountered more bad harvest than good harvest :)

Greatly appreciated your help!

Here is the protocol we use for culturing amniotic fluid (we made it based on AGT Laboratory Manual Book and modified it):

1.  Centrifuge the amniotic fluid sample for 10 minutes at 1000 RPM. Usually we get around 10 - 15 mL of amniotic fluid per sample. 

2.  Discard the supernatant and leave about 1 mL or as much as the pellet

3. Wash the cells by adding 2 mL HBSS to cover the cell surface

4. Homogenize HBSS in the flask by gently shaking the flask

5. Discard HBSS

6. Add 3 mL of AmnioMax, spread the media to cover the flask surface by gently shaking the flask

7. Resuspend the cells by adding 3 mL of AmnioMax

8. Transfer the cell suspension to the flask, homogenize the suspension to cover the flask surface

9. Close the flask tightly and place it in the incubator (37°C, 5% CO2)

10. Incubate and observe again after 4 days

11. Repeat steps 10-16 until the cells are 80% confluent

(Usually our culture goes through 4 media changes, so around 12-16 days to reach 80% confluency. We use T-25 flask. We are also working on how to shorten the culture time)

(We use HBSS to wash the cells starting from the 3rd media change, should we use it from the first media change?)

Harvest and Staining Procedure

KCl and Carnoy's solutions are prepared fresh on the day of work

The prepared KCl solution is placed in a water bath at 37°C

The prepared Carnoy's solution is stored at a cold temperature (2-8°C)

1. 30 minutes before harvesting time, add 150 µL of colchicine, continue incubation for 30 minutes
2. After incubation, transfer the media and colchicine to a labeled conical tube (patient's name, patient's lab number, harvest date)
3. Wash the cells in the flask by adding 2 mL HBSS, transfer to a conical tube
4. Add 2 mL of 0.2% TripleExpress, incubate for 3 minutes in the incubator until the amniotic cells detach from the flask. Gently tap or shake the flask to help the cells detach from the bottom surface of the flask
5. Add 2 mL of AmnioMax, wash and collect the cells that have detached from the flask surface, then transfer to a conical tube
6. If there are still cells that have not detached from the flask, repeat steps 4 and 5 with an incubation time of 5 minutes for 0.2% TripleExpress
7. Centrifuge at 1000 RPM for 10 minutes
8. Discard the supernatant and leave ± 1 mL in the conical tube
9. Resuspend
10. Set a timer for 30 minutes, add 3 drops of warm hypotonic solution, resuspend. Add up to 9 mL
11. Shake vigorously for 10 seconds then incubate in a water bath at 37°C for the remaining 30 minutes
12. Add 5-10 drops of cold fixative solution through the tube wall, resuspend
13. Centrifuge at 1000 RPM for 10 minutes
14. Discard the supernatant and leave ± 1 mL in the conical tube
16. Gently resuspend, add 3 drops of fixative solution through the tube wall, resuspend. Add up to 2 mL, resuspend.
17. Leave at room temperature for 20 minutes
18. Centrifuge at 1000 RPM for 10 minutes
19. Discard the supernatant and leave approximately 1 mL in the conical tube
20. Add 1 mL of cold fixative solution through the tube wall, resuspend gently
21. Add cold fixative solution through the tube wall up to 7 mL
22. Centrifuge at 1000 RPM for 10 minutes
23. Discard the supernatant and leave approximately 1 mL in the conical tube
24. Repeat steps 19-22 twice
25. Add 1 mL of cold fixative solution, resuspend, and then perform dropping 2-3 drops onto the labeled slide
26. The slides are dried at room temperature for 3-4 days.
27. The slide is dipped in a 0.1% trypsin solution for approximately 2 seconds. 
28. Wash the slide in PBS solution with 2-3 dips. 
29. Drop and spread the Giemsa-Wright solution evenly on the slide surface, then let it sit for 2 minutes. 
30. Rinse with slow running water. Dry the slide and then analyze it under a microscope.

[EVANGELIDOU Paola]

Dear Valerie hello,

 

I am sure Maria will respond at some point, but since I had a quick look at your harvesting protocol I thought of sharing my thoughts. You may wish to adjust the time that your cultures are treated with colcemid. In our Lab we add colcemid 2 hours and 45 minutes before harvesting. I am not sure if we are using the same concentration of colcemid though. Ours is Colcemid (10µg/ml)  from Capricorn Cat# COL-H.

 

Kind regards,

 

Paola

 

 

Paola Evangelidou, PhD
Scientist | Cytogenetics and Genomics Department
Academic Staff | The Cyprus Institute of Neurology & Genetics
6 Iroon Avenue, 2371 Ayios Dometios, Nicosia, Cyprus | PO Box 23462, 1683, Nicosia, Cyprus
: +357 - 22 358600 (Switchboard) | : +357 - 22 392726 (Direct Line) | Fax: +357 22 392793 | : pa...@cing.ac.cy | : https://www.cing.ac.cy

  

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From: 'Valerie Tio (2402119161)' via Cytogenetics methods and trouble-shooting Forum <cytogenetics-methods...@googlegroups.com>
Sent: 14 August 2024 04:37
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Subject: Re: Elongating Chromosomes for GTG-banding without Toxic Chemicals

 

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[Cytogenetics methods and trouble-shooting Forum]

Hi Valerie,

Welcome to the forum and thanks for sharing your experience with amniotic fluid culturing for GTG-banding. It's great to see someone tackling this challenge!

Inconsistent harvest results are indeed a common frustration in this area. Your description of the issue sounds familiar to many in the field. The ratio of AmnioMax Basal to supplement you're using seems standard, but there might be other variables influencing your outcomes.

Thank you for sharing images of your metaphases as its incredibly helpful for troubleshooting.

I encourage other forum members with experience in this area to share their insights and suggestions.

I remind all members that this forum is still very active indeed!

Moderator

[Valerie Tio (2402119161)]

Hi Paola. Thank you for the advice. It seems that I do have to optimize the incubation time for colcemid since my harvest often results in low metaphase, indicating low mitotic index. We are using the same concentration of colcemid (10µg/ml), although the one I am using is from karyomax.

Thanks for your advice, greatly appreciate it! 

Valerie 

[EDUARDO DE LA ROSA]

Hello Valerie:
For amniotic fluid samples and products of conception we use Colchicine 10mg/ml, we prepare it from  colchicine SIGMA C - 9754. We add 75 ul for 20 minutes and it functions ok. We do it for many years and we haven't had any problem with the prenatal cultures.

Blgo. Eduardo De La Rosa
Clínica Alemana de Santiago - Chile

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[Valerie Tio (2402119161)]

Hi! Thank you for the insight. I have two questions:

1. How many mL of culture medium do you typically use in a flask when adding 75 µL of colcemid for 20 minutes?
2. How long does it usually take for your cultures to reach 80% confluency?
3. What media do you use? 

Thank you for your help!

Regards,

Valerie 

[maria mouriki]

Hello again! 

You protocol sounds good and I am not a big fan of making huge changes in a protocol that has been working up until now. My ideas are the following:

1. In order to maximize the number of metaphases and get longer chromosomes I suggest using smaller concentration concentrations of colcemid for longer periods. In addition to colcemide we use BRDU solution. Our flasks have 5ml of medium and in that I add 40μl of colcemide (10μg/ml) and 90μl of BRDU (which I make by dissolving 100mg BRDU powder in 33ml of water for injection) and leave overnight 12-16 hours.

2. Optimize/change/experiment with your slide making conditions. I have noticed that in our lab, making preps are difficult during summer time (mitotic index and length) and I have to improvise by making chromosome preps on wet paper or dry over a heater (yes, that's correct! we use a heater when humidity is too high or too low even if the temperature is high).

3. One step which I don't see in your protocol and I think is worth trying, as we are talking about flasks and not in situ harvest on coverslips, is to treat your cells with TRYPSIN/EDTA when the colonies are nice and active, also known as subculturing. This will definitely increase your mitotic index. When my colonies are ready (usually 10-12 days) I remove the medium, wash quickly and gently with 1 ml T/E, pour off that, add another ml T/E, swirl a bit, remove it and let the flask rest horizontally in the incubator for a few minutes (5-10') so as the colonies to break apart and cells to float free in the flask (will always need some tapping on the bench in order to get all the cells to detach from the flask). Afterwards I add 5ml of medium and leave in the incubator UNTIL I see nicely dividing cells all over the place (it takes 1-2 days to do so). (Think about it... in a tight colony only the cells at the periphery are usually active and take up colcemide. But when you spread the cells all over the flask you get more actively dividing cells taking up colcemide, so higher mitotoc index).  Then I add colcemide/BRDU. [By the way, I really like to leave cells in their peace and quiet at least for a week after setting up. They are more resilient than we think. Afterwards I check them every 2 days BUT do not change the medium for another week, where by until then I have already subcultured them. 5ml of medium are enough for a week (no need to try that, just sharing my experience)].  The trypsin/EDTA solution I use is ''Trypsin-EDTA 1X in PBS w/o Calcium w/o Magnesium w/o Phenol Red'' by BIOSERA.

I think that's all I can think of for helping you. If you try any of that or other that people suggested already please do inform the forum so the knowledge is shared.... (failure of a protocol is also knowledge...) 

Keep up the good work! 

Maria Mouriki

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[EDUARDO DE LA ROSA]

Hi, Valerie:

We don't use T- flask, we use petri dishes corning 60 x 15mm and add 75ul of COLCHICINE 10 mg/ml for 3ml of medium. We harvest our cultures in approximately 9 - 11 days. We use 3 petri dishes with 3 types of mediums: A) Amniomax C100, supplement and basal. B) Amniomax II, medium complete and C) AmnioPrime from Capricorn.

To prepare the colchicine we use H2O bidistilled, 0.1 gr of colchicine in 10ml of water---- 10mg/ml.

Eduardo De La Rosa R.

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[Vytautas Šliužas]

Vytautas Šliužas reacted to your message:

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From: cytogenetics-methods...@googlegroups.com <cytogenetics-methods...@googlegroups.com> on behalf of maria mouriki <mendel...@gmail.com>
Sent: Wednesday, August 21, 2024 2:22:26 PM
To: cytogenetics-methods...@googlegroups.com <cytogenetics-methods...@googlegroups.com>

Subject: Re: Elongating Chromosomes for GTG-banding without Toxic Chemicals

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