I am trying to optimize ADCC for HIV infected cells. I am labeling my targets with CFSE and any other membrane or nuclear dye. My effectors are unlabeled. The problem is the second dye, no matter what is that, gets transferred to unlabeled effectors. So how can I stop the transfer process? Or what kind of dye I should use?
Can you tell me the purpose of labelling with a second dye and what sort of dyes you have used? Shouldn't CFSE rejection be sufficient to tell you how much killing has happened? We have used cell trace violet, CFSE and cell proliferation dye efluor670 in combination for CD8+ T cell killing assays in vivo without the issues you are referring to.
The purpose of second dye is to identify my target population. If CFSE gets leaked out, lysed cells have same CFSE position as my effectors. So a second dye is essential we thought.
I used PKH26 and Deep red. Both are membrane dye. Now I have tried vybrant ruby, its a nuclear dye.
Every time when I incubate, unlabeled cells take up the dye. So instead of having two population (negative and positive) in second dye channel, I see one population and that is dimmer than targets only.
Due to the transfer of membrane dyes between target and effector cells, they can be used in evaluating killing assays.
Find this section in the attached file:
"Tracking the Effects of Anti-Tumor Immunotherapy
One of the earliest applications of membrane dyes was as a nonradioactive probe for tracking NK cell killing of tumor
targets5. As recently reviewed by Zaritskaya6, variations on this theme have expanded exponentially in the last decade.
Membrane dye labeling of target cells and/or effector cells combined with multicolor flow cytometry continues to provide
detailed insight into signaling pathways, mechanism of killing, and other factors affecting cytotoxic efficacy..."
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