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Cell clumping: dead cells?

Flow live culture transport

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#1 spookyj1983

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Posted 14 October 2013 - 07:10 AM

Hi,

 

I tried an experiment where I took an adherent cell culture (grown on 10 cm plates) out of an incubator, drove them to another facility (about 1.5 hours away), administered a treatment, trypsinized, and permeablized and fixed them with PFA and ethanol before driving them back and analyzing through flow cytometry. In all, they were outside of an incubator and sloshing around for about 5 hours (although I had a hot plate and kept them at 37 degrees when they were out of the car).

 

Upon staining them with PI and prior to passing them through flow, the cells all became clumped together and the flow yield was very low.

 

My thought is that the stress outside of the incubator may have killed a significant proportion of the cells, and that the DNA from the lysed cells is causing the remaining population to clump. The next things I am thinking of trying:

 

1. Cutting down on the time that cells are outside of the incubator before they are treated and fixed (to minimize the number of dying cells)

2. Considering using Hepes or another buffered media (as pH may be responsible for the stress that's killing the cells)

3. Using DNAse or Accumax to decrease the cell clumping after fixation.

 

Number 3 makes me a little nervous because the assay that I am running after fixation is DNA based, and I'm worried that adding DNAse to the buffer will just chew up the DNA in these permeabilized cells.

 

Does anyone have any suggestions about how to minimize cell death and clumping in this set up?

 

Thanks!



#2 antitoxin

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Posted 14 October 2013 - 12:12 PM

perhaps (& perhaps) centrifuge metod in 1200 rpm 7min is helpful



#3 bob1

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Posted 14 October 2013 - 12:58 PM

I would have thought that before trypsinising the cells, you would have washed them in PBS etc, which should remove most of the dead/floating cells.  Once they are fixed, there shouldn't be any further lysis of the cells (unless your fixing is incomplete), so the transport to or from your facility should not be the problem.

 

It is much more likely that the cells are clumping while trypsinising them.



#4 Tabaluga

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Posted 15 October 2013 - 05:31 AM

What does your FACS Buffer consist of ? BSA for instance generally helps prevent cell clumping.


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
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Comme la nuit se fait lorsque le jour s'en va.

 


#5 spookyj1983

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Posted 15 October 2013 - 09:01 AM

I would have thought that before trypsinising the cells, you would have washed them in PBS etc, which should remove most of the dead/floating cells.  Once they are fixed, there shouldn't be any further lysis of the cells (unless your fixing is incomplete), so the transport to or from your facility should not be the problem.

 

It is much more likely that the cells are clumping while trypsinising them.

 

 

What does your FACS Buffer consist of ? BSA for instance generally helps prevent cell clumping.

 

Thanks for the replies. bob1, I think you may be right about this. Because I don't have an incubator at the facility, I was worried that I needed to allow the trypsin to sit for longer at room temperature, so I allowed the trypsin to sit for 15 mins. After discussing with others, people generally feel this is way too long, even for room temp.

 

I'm going to let trypsin sit on the plate and watch it continuously with the microscope until the point the cells begin to round. This will be the time point I use going forward. Hopefully this will cut down on the clumping.

 

Tabaluga: thanks for the tip, the buffer does in fact have BSA in it.

 

antitoxin: Not sure how longer or faster spinning would help with the cell clumping issues, could you elaborate?

 

Thanks again!



#6 bob1

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Posted 15 October 2013 - 11:47 AM


Thanks for the replies. bob1, I think you may be right about this. Because I don't have an incubator at the facility, I was worried that I needed to allow the trypsin to sit for longer at room temperature, so I allowed the trypsin to sit for 15 mins. After discussing with others, people generally feel this is way too long, even for room temp.

 

I'm going to let trypsin sit on the plate and watch it continuously with the microscope until the point the cells begin to round. This will be the time point I use going forward. Hopefully this will cut down on the clumping.

That is a good idea.  I never leave trypsin on for a defined time, just until the cells start to lift off.  For most cell lines this will be between 3-7 min at room temp (I almost never use a incubator either), so I think you will be OK with what you are going to do.



#7 spookyj1983

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Posted 04 November 2013 - 12:43 PM

 


Thanks for the replies. bob1, I think you may be right about this. Because I don't have an incubator at the facility, I was worried that I needed to allow the trypsin to sit for longer at room temperature, so I allowed the trypsin to sit for 15 mins. After discussing with others, people generally feel this is way too long, even for room temp.

 

I'm going to let trypsin sit on the plate and watch it continuously with the microscope until the point the cells begin to round. This will be the time point I use going forward. Hopefully this will cut down on the clumping.

That is a good idea.  I never leave trypsin on for a defined time, just until the cells start to lift off.  For most cell lines this will be between 3-7 min at room temp (I almost never use a incubator either), so I think you will be OK with what you are going to do.

 

Hi everyone,

 

So another set back. I simulated the experiment with gentle tryspinization and got encouraging results; the cells survived and there was no clumping. However, when I tried to replicate at the facility that was 1.5 hours away, I again got cell clumping after treating with primary and secondary antibody and staining with P.I.

 

So now, it is back to square one.

 

Some thoughts and questions:

 

1. When I simulate the experiment, the cells are fixed with ice cold ETOH and then placed directly into the -20. At the other facility, they are fixed with ice cold ETOH, sit on half melted ice for 1 hour, and then go into the -20. Could this additional time when they are sitting on ice and not in -20 be enough to cause significant clumping?

 

2. My primary antibody is against a intranuclear protein, and my secondary is goat anti-mouse flourescent. Could the antibodies be causing the clumping? (I didn't go treat with antibody during my simulation).

 

3. Does anyone have experience using Accumax or other anticlumping reagents AFTER staining but BEFORE sending through flow? My protein of interest associates with DNA and I am worried that the DNAse will end up affecting things, especially with permeabilized cells.

 

Thanks!







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