Homemade reagents vs fluidigm kit.
Posted: Mon Mar 21, 2022 3:06 pm
HI CYTOF community,
I started using cytof few month ago and now I’m moving to more « real » experiments involving barcoding and staining of multiple samples.
I have two main questions that are linked :
1) I curently use the Cell-ID Palladium barcoding kit from fluidigm allowing barcoding of up to 20 samples. This kit is insanely expensive. Do you guys usually do your own barcodes or use commercial kit ? And if you use homemade barcode do you usually use the recipe from Zunder and al. ?
2) I usually don’t permeabilize my samples and instead use the fix and perm buffer contained in the kit. When I do my stain, some time antibodies works and some time they don’t work (same antibodies, same type of sample) so I was wondering if permeabilization might be the problem. As it is impossible to know what is in the fix and perm buffer from fluidigm, I was wondering if it could be a transient permeabilization (saponin like) and that when I add my antibodies, depending on how « intact » the cells are sometime the Ab goes in and sometime don’t go in.
Thanks a lot in advances for your help, have a good day.
Simon
I started using cytof few month ago and now I’m moving to more « real » experiments involving barcoding and staining of multiple samples.
I have two main questions that are linked :
1) I curently use the Cell-ID Palladium barcoding kit from fluidigm allowing barcoding of up to 20 samples. This kit is insanely expensive. Do you guys usually do your own barcodes or use commercial kit ? And if you use homemade barcode do you usually use the recipe from Zunder and al. ?
2) I usually don’t permeabilize my samples and instead use the fix and perm buffer contained in the kit. When I do my stain, some time antibodies works and some time they don’t work (same antibodies, same type of sample) so I was wondering if permeabilization might be the problem. As it is impossible to know what is in the fix and perm buffer from fluidigm, I was wondering if it could be a transient permeabilization (saponin like) and that when I add my antibodies, depending on how « intact » the cells are sometime the Ab goes in and sometime don’t go in.
Thanks a lot in advances for your help, have a good day.
Simon