I guess the only way to get rid of the magnetic particles is to wait until the cells have taken them up and digested them, if that is possible at all. Considering that the beads are bound to antibodies which you use to stimulate your cells there is no chemical reaction you could use that wouldn't at the same time kill your cells and hence be counterproductive to you expansion.

Consult a published report and try it to find out how best it works in your hand.

Then you should not remove the beads. I don't have any  problems when I culture magnetic isolated cells, without removing the magnetic beads.

Thank you all. Ijsbrand i have thought of that. Do you think i can leave them in flow cytometry as well?

Signal wise it should be possible to analyse your cells with the magnetic particles, you will just need to adjust our scatter and maybe fluorescence settings accordingly. Depending on the size of the particles and the type of flow cytometer your are using (hydrodynamic focusing or not) you might run into problems with clogging the flow cytometer. Diluting your sample should help.

Not so sure about flow sorting - you might have more problems as you use charges for sorting and a magnetic particle will influence the system negatively.

I used flowcytometry without any problems (as far as I know). Dilution is the key I think.

Dear Oriana, 

May I ask exactly which product you are using? Since you mention Expander is it the Dynabeads CD3/CD28?

kind regards

ketil

Yes it is

Time is critical here. The first 2-3 days the beads are very adhered to the cells in the activation process. After 2-3 days the bead-bound cells can be pipetted thoroughly to remove the beads from the cells, applied to the magnet to remove the beads and then use the bead-free cells in the flow.  

I agree with Berit. 

This is due to the fact that the cytoplasmic tails of the CD3 subunits and TCR interact with cytosolic-signalling proteins. In this cytosolic part of the complex several types of internalization signals are located which can be divided into two different groups.

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 beads are binding to the CD3-CD28 the complex will actively be internalized and degraded. 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. If the beads are not removed from the media they can re-bind to the TCR-CD3 complex when they re-appear at the plasma membrane.

The T cells when activated properly will form aggregates around the beads (spherical structures) indicating the stimulation is very good. The difference in patient samples may be due to variation in cell status, i.e. how easy to activate.

With good stimulation it is very hard to break the aggregates up the first 1-3 days. However, the TCR/CD3-complex will be down-regulated on the cells upon activation and then the beads will fall off very easy.

hope this helps

ketil

Thanks! You have been very helpful!

Dear Oriana, in case yout still face the problem. please try our CD3 pluriBeads. Detaching works within 10 minutes at RT. Releasing ist about 99% and you can use the pluriBeads directly in whole blood.

BW

Michael