I wouldn't like to have PFA in a well next to live cells in an incubator.... It might be paranoia but I worry about it affecting the quality of the other cells in the experiment. When I've had similar experiments previously I've simply moved the coverslips into a separate 24 well plate at each time-point and processed these cells as normal.
I wouldn't like to have PFA in a well next to live cells in an incubator.... It might be paranoia but I worry about it affecting the quality of the other cells in the experiment. When I've had similar experiments previously I've simply moved the coverslips into a separate 24 well plate at each time-point and processed these cells as normal.
Thank you! The only thing I worry about it that the cell dynamics might change during this time if I increase the temperature. I assume by adding ice cold PFA this will reduce cell processes to give me a rough idea of cell dynamics at my given time points?
Thank you! The only thing I worry about it that the cell dynamics might change during this time if I increase the temperature. I assume by adding ice cold PFA this will reduce cell processes to give me a rough idea of cell dynamics at my given time points?
Hi Philip - yes, I would usually do that too, but I'm using a special 96-well imaging plate so I'm stuck using the same plate for fixing wells at all time points! Thank you for the advice though.
Hi Philip - yes, I would usually do that too, but I'm using a special 96-well imaging plate so I'm stuck using the same plate for fixing wells at all time points! Thank you for the advice though.
in my experience, 37 D for 10 min is optimal for mitochondrial study, as mitochondrial is sensitive to temperature, RT fixation will give quit different pattern of mitochondrial net in cells.
in my experience, 37 D for 10 min is optimal for mitochondrial study, as mitochondrial is sensitive to temperature, RT fixation will give quit different pattern of mitochondrial net in cells.
Yes, you can fix at 37C with PFA. Fix for 5-20 mins (10 min would be a good average).
Good luck!
Yes, you can fix at 37C with PFA. Fix for 5-20 mins (10 min would be a good average).
Good luck!
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I wouldn't like to have PFA in a well next to live cells in an incubator.... It might be paranoia but I worry about it affecting the quality of the other cells in the experiment. When I've had similar experiments previously I've simply moved the coverslips into a separate 24 well plate at each time-point and processed these cells as normal.
I wouldn't like to have PFA in a well next to live cells in an incubator.... It might be paranoia but I worry about it affecting the quality of the other cells in the experiment. When I've had similar experiments previously I've simply moved the coverslips into a separate 24 well plate at each time-point and processed these cells as normal.
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Thank you! The only thing I worry about it that the cell dynamics might change during this time if I increase the temperature. I assume by adding ice cold PFA this will reduce cell processes to give me a rough idea of cell dynamics at my given time points?
Thank you! The only thing I worry about it that the cell dynamics might change during this time if I increase the temperature. I assume by adding ice cold PFA this will reduce cell processes to give me a rough idea of cell dynamics at my given time points?
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VOTE
I do that too. Just shorten the time as the chemical act faster at elevated temperature. You can use cold PFA (i.e., just removed from the fridge).
I do that too. Just shorten the time as the chemical act faster at elevated temperature. You can use cold PFA (i.e., just removed from the fridge).
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Hi Philip - yes, I would usually do that too, but I'm using a special 96-well imaging plate so I'm stuck using the same plate for fixing wells at all time points! Thank you for the advice though.
Hi Philip - yes, I would usually do that too, but I'm using a special 96-well imaging plate so I'm stuck using the same plate for fixing wells at all time points! Thank you for the advice though.
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VOTE
I thought that something like that might be the case as I was writing my answer. Good luck!
I thought that something like that might be the case as I was writing my answer. Good luck!
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in my experience, 37 D for 10 min is optimal for mitochondrial study, as mitochondrial is sensitive to temperature, RT fixation will give quit different pattern of mitochondrial net in cells.
in my experience, 37 D for 10 min is optimal for mitochondrial study, as mitochondrial is sensitive to temperature, RT fixation will give quit different pattern of mitochondrial net in cells.
More
VOTE
Hello
We usually fixed tissues at 37 C for 10 mins
Good Luck
Hello
We usually fixed tissues at 37 C for 10 mins
Good Luck
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VOTE