Fixation and Ir Intercalactor staining
Hej all,
I am totally new to CyTOF and we are setting up our first panel. I have a few questions about the general protocol and how we could shorten/combine things. I guess a lot of users here have tried these things. We are running the Helios system.
1. Has anybody tried to stain Ir Cell-ID at the same time as the intracellular antibodies in the Perm buffer of the FoxP3 kit?
2. Could you stain Ir Cell-ID in “% or 4% fixing buffer for 30 min after you have used the FoxP3 staining kit?
3. There are a lot of different protocols fixing with either 2% or 4 % PFA. Is there any disadvantage/advantage of one over the other?
4. What about leaving the cells in 4% PFA with Ir Cell-ID overnight in the fridge? Do you lose many of the cells or is it okay?
5. Any experience with staining CD45 barcoding while staining with Rhodium-103 for live/dead discrimination?
Thanks for your help!
Cheers, Louis
I am totally new to CyTOF and we are setting up our first panel. I have a few questions about the general protocol and how we could shorten/combine things. I guess a lot of users here have tried these things. We are running the Helios system.
1. Has anybody tried to stain Ir Cell-ID at the same time as the intracellular antibodies in the Perm buffer of the FoxP3 kit?
2. Could you stain Ir Cell-ID in “% or 4% fixing buffer for 30 min after you have used the FoxP3 staining kit?
3. There are a lot of different protocols fixing with either 2% or 4 % PFA. Is there any disadvantage/advantage of one over the other?
4. What about leaving the cells in 4% PFA with Ir Cell-ID overnight in the fridge? Do you lose many of the cells or is it okay?
5. Any experience with staining CD45 barcoding while staining with Rhodium-103 for live/dead discrimination?
Thanks for your help!
Cheers, Louis