Hi, your chromosomes are in late metaphase. This is a sign that they are already beginning to separate chromatids in order to enter the next phase of division-anaphase.I suppose that the division clock of mitosis is important.

How long do you keep the cell culture in colchicine? How much colchicine do you add per ml of medium? Do you change the medium 24 hours before? If yes, how many hours after changing the medium do you add colchicine?

Your G-strings are also unsuccessful. Fuzzy and unreadable. You should change something in the procedure to get a higher quality of analyzed chromosomes.

Best, Sylwia

Hi Sylwia Gawlik-Zawiślak

Thanks for your feedback! I appreciate your insights.

You're right, the late metaphase spread is something I've been trying to optimize. Here's a breakdown of my current protocol:

1. Colcemid concentration: I’m using 150 microliters of Colcemid (10mg/mL) per 3 mL of medium.

2. Incubation time: I've experimented with different Colcemid incubation times (30, 60, 90 minutes) and found that 60-90 minutes gives me the most metaphases.

3. Media change: Yes, I do a media change 24 hours before adding Colcemid and harvesting by reculture/passaging the culture into 2 different flask.

Regarding the G-banding, I agree that the quality isn't ideal. I'm troubleshooting that part of the process as well. Any suggestions for improving the banding resolution would be very welcome!

Do you think adjusting the Colcemid concentration or incubation time further could help with the late metaphase issue?

Thanks again,

Karina

Finding a few late metaphases across the slide is normal. If they’re all like that, the issue likely derives from the culture conditions and colchicine timing. How was the cell growth? How many clones did you have at the time of harvest? You mention that 60–90 minutes of colchicine give you more metaphases, but are they well-condensed? The goal is not to obtain as many metaphases as possible but to have a sufficient number of good-quality metaphases that allow you to perform the analysis according to the guidelines. Try reducing the colchicine exposure to 45 minutes and ensure that there is a sufficient population of dividing cells when you block the cell cycle. Regarding banding, if you don’t start with a good preparation, it will never turn out well.

Regard, Filomena

Hi,

Try to add colchicine 22 h after changing the medium. Try to keep time.

It seems to me that you have too high concentration of colchicine. We use 0.1ml of colchicine (Gibco by Life Technologies) per 5ml of medium, incubate 90 min at 37 C- sometimes 75 min.

We trypsinize from the cell medium. We use hypotonic shock with 0.078 M KCl (sometimes we use OHNUKI buffer), incubate 30 min at 37 C. Then we start fixation with Methanol: glacial acetic acid 3:1. We repeat the operation 3x.

Regards,

Sylwia

The process of obtaining striations on chromosomes is a difficult art. You need to have well-chosen chemical reagents.

We have each patient's trypsinization time and G-band- staining selected individually.

Hi Filomena Mottola ,

Thanks again for your input. To answer your questions about the culture conditions:

- Cell growth: I typically passage the cells after 12 days of culturing, when they're nearing 80% confluency and due for their third media change. I usually harvest from the first passage, or at most the second. I started with 10 mL of amniotic fluid (the other 10 mL was used for microarray).

- “Droplets": I've attached an image of the culture at the time of harvest. I look for those "droplets" as an indicator of dividing cells.

While I think the metaphases are generally well-condensed (long enough to be karyotype and non-overlapping), I agree that the split chromatids are a problem. I've attached a picture of a representative karyotype.

To show that the banding technique itself isn't the main issue, I've also included a karyotype where some chromatids are split, but the banding is still clear enough for analysis. However, I acknowledge that the banding isn't always perfect; some chromosomes appear more gray and black, lacking distinct bands. I'm still working on getting consistently good banding across all preparations.

I'll try reducing the colchicine exposure to 45 minutes and see if that helps.

Do you have any other suggestions based on the images? Perhaps something about the culture conditions or harvesting technique could be contributing to the splitting?

Would you mind taking a look at my culture protocols as well? I'm wondering if there might be something specific in my procedures that could be contributing to this issue.

Thanks,

Karina