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Fixation after or before staining?


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#1 sabi67

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Posted 13 August 2009 - 08:14 AM

Hi every one,

I have to do some flow cytometry on 2 different experiments on mice. The mice from each expriments won't be sack the same day but cells are stained with the same antibodies, so I was wondering if I could use the same compensation controls for samples of the 2 different experiments. I'm not quite sure it's possible, because in this case, half the samples will not be stained at the same time than my compensation controls.
To avoid this, I'm thinking to fix my cells in formaldehyde before I stain so I could keep cells from the first experiment until I have cells from the second one ready (fixed before staineing too). I'm just wondering if the fixation prior to staining can modify my staining, I'm using antibodies againt surface proteins?
Thank you for any help or suggestion.

#2 almost a doctor

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Posted 13 August 2009 - 08:31 AM

Hi every one,

I have to do some flow cytometry on 2 different experiments on mice. The mice from each expriments won't be sack the same day but cells are stained with the same antibodies, so I was wondering if I could use the same compensation controls for samples of the 2 different experiments. I'm not quite sure it's possible, because in this case, half the samples will not be stained at the same time than my compensation controls.
To avoid this, I'm thinking to fix my cells in formaldehyde before I stain so I could keep cells from the first experiment until I have cells from the second one ready (fixed before staineing too). I'm just wondering if the fixation prior to staining can modify my staining, I'm using antibodies againt surface proteins?
Thank you for any help or suggestion.


Fixing is definitely going to affect the surface of your cells, and therefore your staining, to the point that it might not work (you might end up with false results).

I'd recommend you stain your cells and then fix. Keep them in the fridge until you get the 2nd batch, stain, fix and analyse them all at the same time.

How many colours are you using? compensation controls are only a few extra tubes, can you not just prepare them again on the 2nd day?

#3 sabi67

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Posted 13 August 2009 - 08:55 AM

Hi every one,

I have to do some flow cytometry on 2 different experiments on mice. The mice from each expriments won't be sack the same day but cells are stained with the same antibodies, so I was wondering if I could use the same compensation controls for samples of the 2 different experiments. I'm not quite sure it's possible, because in this case, half the samples will not be stained at the same time than my compensation controls.
To avoid this, I'm thinking to fix my cells in formaldehyde before I stain so I could keep cells from the first experiment until I have cells from the second one ready (fixed before staineing too). I'm just wondering if the fixation prior to staining can modify my staining, I'm using antibodies againt surface proteins?
Thank you for any help or suggestion.


Fixing is definitely going to affect the surface of your cells, and therefore your staining, to the point that it might not work (you might end up with false results).

I'd recommend you stain your cells and then fix. Keep them in the fridge until you get the 2nd batch, stain, fix and analyse them all at the same time.

How many colours are you using? compensation controls are only a few extra tubes, can you not just prepare them again on the 2nd day?


Thank you. I'm using 3 colors. That's true I could just do compensation controls again the 2nd day but I try to take as less cells as possible for this staining because I keep cells I don't use for cell sorting and the more cell I have back, the best it is for me.
Will I have a lot of dead cells if I keep them one night in the fridge? Would you keep them in PBS or media?

Thank you

#4 bob1

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Posted 17 August 2009 - 04:18 PM

Day one: stain, fix, store at 4˚C
Day two: stain, fix, run both sets of samples.




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