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I'm getting fried-egg looking cells... What should I do??


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#1 sc_queen

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Posted 20 November 2010 - 10:46 AM

I have attached a picture of my cells here... We have received three SCC cell lines from overseas on Monday, Nov. 15. All of them looked fine, but this one (established from oral squamous carcinoma) started to develope what look like includsion bodies. The medium the cells are immersed in is RPMI1640 + 10% FBS, and the cells were shipped in culture, not frozen. I replaced the media yesterday with a fresh media containing a new batch of serum, but it doesn't seem to help... Is there anything else I can do to rescue my cells? Or is it possible at all??

Any help would be greatly apprecited.

Catherine

Attached Thumbnails

  • OECM1_20101120_1.jpg


#2 bob1

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Posted 20 November 2010 - 01:33 PM

Those cells with the vacuoles will not recover, they are fully senescent. The other cells look OK to me. It is fairly normal to get some cell death after transportation as a live culture, as they are usually subjected to temperature changes, hypoxia, etc.

#3 sc_queen

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Posted 20 November 2010 - 05:44 PM

Those cells with the vacuoles will not recover, they are fully senescent. The other cells look OK to me. It is fairly normal to get some cell death after transportation as a live culture, as they are usually subjected to temperature changes, hypoxia, etc.


Thanks so much for your reply, Bob. It would be great if you are right (i.e. the other cells are fine), but the cell density doesn't seem to have increased over the past two days and there a lot of dead cells floating in the medium... Maybe they have stopped dividing?? Do you think it might be a good idea to split them 1:2 and see how they grow? They are now ~75% confluent.

Thanks!

#4 bob1

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Posted 21 November 2010 - 04:22 PM

Did you keep any of the medium they were transported in? If so, try filtering some of that, remove the medium you currently have on the cells and add the filtered medium. This is basically a conditioned medium, which should help them grow.

It could be that your cells are contact inhibited, so you could try splitting them, which should make them grow again.

Are you growing the cells in the same type of medium as the people you got them from?

#5 sc_queen

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Posted 21 November 2010 - 09:29 PM

Did you keep any of the medium they were transported in? If so, try filtering some of that, remove the medium you currently have on the cells and add the filtered medium. This is basically a conditioned medium, which should help them grow.

It could be that your cells are contact inhibited, so you could try splitting them, which should make them grow again.

Are you growing the cells in the same type of medium as the people you got them from?


Hi Bob - No, I didn't keep the original medium. I split them into two flasks today. The cells are growing in the same medium (i.e. the same catalog number provided by the sender). By the way, I noticed that there are a few white things(?) attached to the bottom of the flask. I think might be contaminated... Mycoplasma???

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  • OECM1_20101121_might-be-infection.jpg


#6 bob1

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Posted 22 November 2010 - 12:26 PM

Definitely not mycoplasma - they are largely intracellular and are hard to see down a normal microscope - more likely to be debris on the outside of the flask or some precipitate from the FBS.

The medium you are using should be fine. If splitting doesn't work, try conditioning some medium over a common cell line (i.e. add some fresh medium to a flask of cells, leave for 24 hours), remove medium, filter with 0.2 um filter, and add to your cells.

#7 sc_queen

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Posted 24 November 2010 - 12:14 AM

Definitely not mycoplasma - they are largely intracellular and are hard to see down a normal microscope - more likely to be debris on the outside of the flask or some precipitate from the FBS.

The medium you are using should be fine. If splitting doesn't work, try conditioning some medium over a common cell line (i.e. add some fresh medium to a flask of cells, leave for 24 hours), remove medium, filter with 0.2 um filter, and add to your cells.


Yes, you were right. It turns out I was looking at debris on the outside of the flask. But still, my cells are looking worse every day. Attached are the pictures of another cell line that came from the same lab. The pictures were taken on the 20th, 22nd and 23rd, respectively. I think I should do mycoplasma test (PCR) first thing tomorrow (I should have done this in earlier..). I really hope that the test comes out negative, otherwise, it will be a mess... But regardless of whether or not they were contaminated, I won't be able to use them, as they may have already accumulated mutations, right? If it's not mycoplasma, how can I find out what went wrong? I would hate to see the same thing happen again...

Attached Thumbnails

  • FaDu_20101120.jpg
  • FaDu_201011122.jpg
  • FaDu_201011123.jpg

Edited by sc_queen, 24 November 2010 - 12:14 AM.


#8 rhombus

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Posted 24 November 2010 - 08:09 AM


Definitely not mycoplasma - they are largely intracellular and are hard to see down a normal microscope - more likely to be debris on the outside of the flask or some precipitate from the FBS.

The medium you are using should be fine. If splitting doesn't work, try conditioning some medium over a common cell line (i.e. add some fresh medium to a flask of cells, leave for 24 hours), remove medium, filter with 0.2 um filter, and add to your cells.


Yes, you were right. It turns out I was looking at debris on the outside of the flask. But still, my cells are looking worse every day. Attached are the pictures of another cell line that came from the same lab. The pictures were taken on the 20th, 22nd and 23rd, respectively. I think I should do mycoplasma test (PCR) first thing tomorrow (I should have done this in earlier..). I really hope that the test comes out negative, otherwise, it will be a mess... But regardless of whether or not they were contaminated, I won't be able to use them, as they may have already accumulated mutations, right? If it's not mycoplasma, how can I find out what went wrong? I would hate to see the same thing happen again...



Dear sc queen,


It is difficult to tell from the 3 attached photo's but it looks like a contamination to me. You cannot see mycoplasma's down the microscope (bright field or phase contrast) unless you stain the cells with Hoescht, and then you will only see mycoplasma contamination if the culture is masssively contaminated.

You are absolutely right when you stated " I won't be able to use them as they may have already accumulated mutations" ...this is correct for all contaminations.

Try and get a new batch of cells.

Kindest regards.

Uncle Rhombus.

#9 sc_queen

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Posted 24 November 2010 - 03:23 PM



Definitely not mycoplasma - they are largely intracellular and are hard to see down a normal microscope - more likely to be debris on the outside of the flask or some precipitate from the FBS.

The medium you are using should be fine. If splitting doesn't work, try conditioning some medium over a common cell line (i.e. add some fresh medium to a flask of cells, leave for 24 hours), remove medium, filter with 0.2 um filter, and add to your cells.


Yes, you were right. It turns out I was looking at debris on the outside of the flask. But still, my cells are looking worse every day. Attached are the pictures of another cell line that came from the same lab. The pictures were taken on the 20th, 22nd and 23rd, respectively. I think I should do mycoplasma test (PCR) first thing tomorrow (I should have done this in earlier..). I really hope that the test comes out negative, otherwise, it will be a mess... But regardless of whether or not they were contaminated, I won't be able to use them, as they may have already accumulated mutations, right? If it's not mycoplasma, how can I find out what went wrong? I would hate to see the same thing happen again...



Dear sc queen,


It is difficult to tell from the 3 attached photo's but it looks like a contamination to me. You cannot see mycoplasma's down the microscope (bright field or phase contrast) unless you stain the cells with Hoescht, and then you will only see mycoplasma contamination if the culture is masssively contaminated.

You are absolutely right when you stated " I won't be able to use them as they may have already accumulated mutations" ...this is correct for all contaminations.

Try and get a new batch of cells.

Kindest regards.

Uncle Rhombus.


Thanks Uncle Rohmbus. I will try to get a new batch of cells (and will do mycoplasma test as soon as I get it!)




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