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Immunostaining for flow cytometry


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7 replies to this topic

#1 Loris

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Posted 24 June 2009 - 12:40 PM

Hi everyone,

I guess this is a basic immunostaining question. I am preparing cells for flow cytometry. First I'm staining for a pluripotency marker, then I'm staining for BrdU.

I add 100 uL of SM (2%FBS, NaN3 in 1xHBSS), then 80% EtOH and incubate at 4*C for 30 minutes or more. Then I permeabilize with BD cytowash/perm buffer (a saponin based buffer used to permeabilize cells) and then I incubate O/N with an antibody against the pluripotency marker. When I retreive the cells in the morning, the cells become one giant mass that cannot be dissociated into single cells. I can no longer proceed with the rest of my staining.

Does anyone know what could be happening? I would love to know peoples thoughts on this :)

Thank-you in advance!

#2 viobio

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Posted 21 September 2009 - 10:49 AM

Can you try immunostaining with antibody for just an hour rather than overnight? If you have to do it overnight, can you leave out the permeabilization (maybe using a methanol fix 5min at -20 degrees to both fix and permeabilize)? I assume that not poking holes in the cells will keep them happier for a longer incubation.
What pluripotency marker are you using? If it is on the surface of the cells, you could leave out permeabilization, maybe try another fix like paraformaldehyde.

Hi everyone,

I guess this is a basic immunostaining question. I am preparing cells for flow cytometry. First I'm staining for a pluripotency marker, then I'm staining for BrdU.

I add 100 uL of SM (2%FBS, NaN3 in 1xHBSS), then 80% EtOH and incubate at 4*C for 30 minutes or more. Then I permeabilize with BD cytowash/perm buffer (a saponin based buffer used to permeabilize cells) and then I incubate O/N with an antibody against the pluripotency marker. When I retreive the cells in the morning, the cells become one giant mass that cannot be dissociated into single cells. I can no longer proceed with the rest of my staining.

Does anyone know what could be happening? I would love to know peoples thoughts on this :)

Thank-you in advance!



#3 balam12

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Posted 17 October 2009 - 09:27 AM

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i wish you guys will help me......
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#4 basudec1509

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Posted 01 February 2010 - 11:21 AM

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its really nice post......






help

#5 euwing

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Posted 05 February 2010 - 02:18 PM

It might be better try to dissociate the cells to single cells, then perform fix, permeabalization, and stain. All the procedure can be performed in a FACS tube for washing and changing the solutions.

#6 DmitryM

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Posted 06 February 2010 - 02:25 AM

It might be better try to dissociate the cells to single cells, then perform fix, permeabalization, and stain. All the procedure can be performed in a FACS tube for washing and changing the solutions.


It is impossible. Probably EtOH is not cold (at least -20C)

#7 euwing

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Posted 06 February 2010 - 08:04 AM

It might be better try to dissociate the cells to single cells, then perform fix, permeabalization, and stain. All the procedure can be performed in a FACS tube for washing and changing the solutions.


It is impossible. Probably EtOH is not cold (at least -20C)


Hi Dmitry,


Could you please explain why it is impossible? I have been using this method for staining intracellular antigen for suspened cells.

#8 DmitryM

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Posted 06 February 2010 - 01:58 PM

It might be better try to dissociate the cells to single cells, then perform fix, permeabalization, and stain. All the procedure can be performed in a FACS tube for washing and changing the solutions.


It is impossible. Probably EtOH is not cold (at least -20C)


Hi Dmitry,


Could you please explain why it is impossible? I have been using this method for staining intracellular antigen for suspened cells.



Hi ! :)

I have misunderstood:)

You are rigth. I meant that it is impossible to destroy aggregates after fixing. and what they arise during staining (warm or old reagents).




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