I am using CD3/28 Dynabeads to activate mouse primary T cells in vitro, which causes aggregation of my cells.
When I do flow cytometry, I have large aggregates of cells, which I cannot use for gating and subsequent analysis. If I were to use a magnet to remove the beads, I would be selecting for less-activated cells.
Additionally, my experimental treatment causes the cells to disassociate from the beads, thereby confounding my analysis.
Has anyone run into a similar problem? Is there a way to remove the beads without losing a proportion of my cells and biasing my data?
I usually stimulate mouse primary splenocytes with plate bound anti-CD3/CD28 antibodies and get good stimulation without any problems in downstream applications. You would need some optimization steps but once optimized, it works as good as dynabeads and never gave any problems of aggregation, in my experience
We do have aggregate formation but we wash the cells (human PBMCs) in PBS-EDTA (we let them stand in it for ten minutes at room temperature or 370C if you prefer and then do the washing steps. This has helped us a lot for assays like proliferation using CFSE.
There is a product from Life Technologies called Dynabeads Flowcomp which enables detachment of the bead from the target cell. It's based on desthiobiotin-streptavidin interactions, and the bond can be competed out with biotin without damaging the cells. Unfortunately they don't have a CD28/CD3 mouse kit, so the only alternative is to use the Flowcomp flexi kit. With this kit you can label your anti-CD3 and anti-CD28 antibodies with desthiobiotin and then capture them on streptavidin dynabeads. After you activate your T cells, you incubate them in their "release buffer" (mainly biotin) and the beads should fall off the cells and can be removed with the magnet. You can actually look under the microscope and see when they detach. Good luck.
HI Reid! Dynabeads coated with CD3 and CD28 are strongly attached to the cells in the first 3-4 days in the activation/expansion process, thus it is not recommended to remove the cells from the beads during the 3-4 first days. For molecular assays, the cells can be lysed while they are still on the beads.
hi,
The cytoplasmic tails of the T-cell receptor (TCR CD3) complex interact with cytosolic-signalling proteins. In this cytosolic part of the complex several types of internalization signals are located.
Constitutive internalization (without ligand binding):
Its described in the literature approximately 20 Tyrosine based (YXXØ) internalization motifs and 2 di-leucine (SDxxxLL) motifs which will constitutively remove the TCR-CD3 complex from the plasma membrane without ligand binding. As far as I know from the literature the Recycling time is approx 100 min (from plasma membrane to early endosomes and back to plasma membrane) and after 10 such cycles the TCR-CD3 complex is targeted for degradation in lysosomes.
Induced internalization (with ligand binding)
The ligand dependent internalization of the TCR-CD3 complex is an Ubiquitin dependent internalization process. Upon ligand binding ubiquitin is coupled to a certain amino acid in the cytosolic tail of the complex which results in a clathrin dependent internalization of the complex followed by lysosomal degradation. According to the literature it will take approx 72 hours to reach normal level of TCR-CD3 at the plasma membrane after induced internalization.
When the Dynabeads are binding to the CD3-CD28 the complex will actively be internalized and degraded after a while. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the media.
A biological release mechanism!
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