Karyotyping protocol for human IPS cells?

[Anagha Sawant]

Hi all

Could anyone be willing to share their protocol for karyotyping human IPS cells with  Giemsa stain? I have been doing karyotyping for rat and mouse embryonic stem cells using Giemsa-Trypsin banding. Since human IPS cells divide slower than rodent ES cells, I was also wondering about concentration and duration of treatment with colcemid or comparable chemical. 

Thanks in advance for your help.

Regards,

Anagha
--
Life isn't about how
to survive the storm,
                But how to dance in the rain*****

[laevsky ilana]

Hi Anagha,

please see protocol for karyotyping of human ES and IPS. It's adapted for 2D and 3D. Ask me if you need additional information.

Regards

Ilana

 

Protocol for Karyotype Preparation

The values are calculated for one of six-well plates (10 cm²).  Use dividing cells on the third or fourth day post passaging.

 

1.            Change the medium of the examined well with 2 ml fresh ES cell medium.

 

2.             Add 30ml CRA solution (working solution is 1:100).

 

3.            Incubate for 1 h.

 

Alternative method for chromosome resolution improving: 2.      Add 8ml of sterile BrdU solution (working solution is 10-2 M). 3.      Incubate overnight.     Continue with colcemid solution.

 

4.            Add 150 ml colcemid solution.

 

5.            Incubate for 30 min.

6.      Remove medium and collect the cells with trypsin.

7.      Centrifuge 5 minutes at 1,200 rpm.

8.      Aspirate upper liquid and add 5 ml of Hypotonic Solution pre-warmed at 37^oC.

9.     Incubate 30 minutes at 37^oC.

10.    Add, drop by drop, 1 ml of fixer (see below) on the Hypotonic Solution.

11.   Centrifuge 5 minutes at 1,200 rpm.

12.    Aspirate all liquid and add 5 ml of fixer.

13.  Repeat stages 11-12 twice.

14.  On the third repeat, leave the fixer and keep at -20 ^oC. Can be stored for a few months.

 

 

Fixer

1:3 (Acetic acid : Methanol)

Must be kept at -20 ^oC throughout the procedure. Use a fresh fixer for each preparation.

                       

Hypotonic Solution

KCl 0.56g + Na citrate 0.5g/200ml H₂0

 

 

 

 

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[Anagha Sawant]

Thank you so much for the reply. 

What is CRA solution used before colcemid? 

Thank you once again.

Regards,

Anagha

[laevsky ilana]

Hi Anagha,

CRA is Cromosome Resolution Additive from Genial Genetic Solutions Ltd (JL003A). As you can see from the name it's improves chromosomal resolution. The alternative method is with BrDU (bordered). You can collect the cells without CRA or BrdU, only colcemid, but your chromosomes will be shorter.

Ilana

-- Life isn't about howto survive the storm,                But how to dance in the rain*****

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[andrew]

Hello ilana I as well use CRA having replaced our troublesome Ethidium
Bromide with it. I notice in your protocol the concentration of CRA is
wrong...its too high!
I make a working solution by mixing 0.1ml of CRA stock in 0.9ml of
injection water and only use 0.lml of working solution per l0ml
culture.

On Apr 22, 3:03 pm, laevsky ilana <la140...@yahoo.com> wrote:
> Hi Anagha,
> please see protocol for karyotyping of human ES and IPS. It's adapted for 2D and 3D. Ask me if you need additional information.
> Regards
> Ilana
>  Protocol for Karyotype Preparation
>
> The values are calculated for one of six-well plates (10 cm2).  Use dividing cells on the third or fourth day post passaging.
>
> 1.            Change the medium of the examined well with 2 ml fresh ES cell medium.
>
> 2.             Add 30ml CRA solution (working solution is 1:100).
>
> 3.            Incubate for 1 h.
>
> Alternative method for chromosome resolution improving:
> 2.      Add 8ml of sterile BrdU solution (working solution is 10-2 M).
> 3.      Incubate overnight.
>     Continue with colcemid solution.
>
>
> 4.            Add 150 ml colcemid solution.
>
> 5.            Incubate for 30 min.
>
> 6.      Remove medium and collect the cells with trypsin.
>
> 7.      Centrifuge 5 minutes at 1,200 rpm.
>
> 8.      Aspirate upper liquid and add 5 ml of Hypotonic Solution pre-warmed at 37oC.
>
> 9.     Incubate 30 minutes at 37oC.
>
> 10.    Add, drop by drop, 1 ml of fixer (see below) on the Hypotonic Solution.
>
> 11.   Centrifuge 5 minutes at 1,200 rpm.
>
> 12.    Aspirate all liquid and add 5 ml of fixer.
>
> 13.  Repeat stages 11-12 twice.
>

> 14.  On the third repeat, leave the fixer and keep at -20oC. Can be stored for a few months.

>
>
> Fixer
> 1:3 (Acetic acid : Methanol)

> Must be kept at -20oC throughout the procedure. Use a fresh fixer for each preparation.

>
> Hypotonic Solution
> KCl 0.56g + Na citrate 0.5g/200ml H20
>
>
>
>
> From: Anagha Sawant <anagh...@gmail.com>
> To: cytogenetics-methods...@googlegroups.com
> Sent: Thursday, April 19, 2012 8:56 PM
> Subject: Karyotyping protocol for human IPS cells?
>
> Hi all
> Could anyone be willing to share their protocol for karyotyping human IPS cells with  Giemsa stain? I have been doing karyotyping for rat and mouse embryonic stem cells using Giemsa-Trypsin banding. Since human IPS cells divide slower than rodent ES cells, I was also wondering about concentration and duration of treatment with colcemid or comparable chemical.
> Thanks in advance for your help.
>
> Regards,
> Anagha
> --
> Life isn't about how
> to survive the storm,
>                 But how to dance in the rain*****
>
> --
> You received this message because you are subscribed to the Google Groups "Cytogenetics methods and trouble-shooting Forum" group.
> To post to this group, send an email to cytogenetics-methods...@googlegroups.com.
> To unsubscribe from this group, send email to cytogenetics-methods-and-t...@googlegroups.com.

> For more options, visit this group athttp://groups.google.com/group/cytogenetics-methods-and-trouble-shoot....

[laevsky ilana]

Hi Andrew,

According to manufacturer's instruction, we must prepare a fresh working solution once a week. If you working according this, you right about the concentration. I working now in the stem cells and IPS cells center, and, from my experience, it's not justified for our amount of samples to prepare fresh working solution weekly. I tried using it for a long time and it's working good about 1 year!  So I need a higher concentration, than in the "fresh case".

Ilana

> To unsubscribe from this group, send email to cytogenetics-methods-and-trouble-shooting+unsub...@googlegroups.com.

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