In my research I will sort the specific phenotype of CD3+ CD4+ CD28+ NKG2D+ T cells by using the cell sorter machine and after that I will stimulate the NKG2D receptor but there is a big problem here. How can I stimulate the NKG2D receptor if the receptor has been blocked by the antibody that I use to sort the cells?
My suggestions
1. I have done Treg suppression assays where I used to do cell sorting followed by stimulation with CD3 . You cannot completely saturate the antigens using antibody. Still phenotypes can be ascertained.
2. To easen your problem why dont you try MACS sorting using REAlease Immunomagnetic Separation Technology from miltenyi@ . I have done MACS sorting multiple times and I had planned use of this technology , but I did not used .
This is the reason for using negative selection in many cases, to make sure that the antibodies are not bound to the cell. In some cases you can trypsinize but it is really not recommended since you can cleave a lot of receptors from the cell membrane...... A good possibility is if yo have an anti-idiotipic antibody, but usually this is not the case
Hi Rian,
This might help.
After FACS sorting, incubate your cells with 10 ml of complete cell culture medium at 370 C for 10-15 mins, shake gently and centrifuge. Dissolve the pellet again with 10 ml of complete cell culture medium and incubate at 370 C for 10-15 mins, shake gently and centrifuge. Repeat the step for three more times (Total five times).
With each washing step as mentioned above, you will disrupt the association constant of the antibodies and thus eliminate some of the antibodies at the supernatant in each washing step.
Note: Do not forget to pre-heat your medium to 37 degree to use in each step.
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