Hello Sergio!

I our lab we use a MACS based kit, and for major yield we isolate from mouse spleens (in case you want to obtain murine Th Cells populations). We obtain pretty solid amount of cells for reproducible differentiation protocols.

As you point, we use these enriched CD4+ cells to assess the modulation of Th1, Th2 or Th17 differentiation under different treatments, being usual readings the cytokine production (TNFa, INFg, IL23, IL17, IL6, IL10 etc...), RT-qPCR of relevant genes (TBET, GATA3, FOXP3 etc.) or flow cytometry as you point. Also, you have the possibility to see this modulation during the differentiation or in mature differentiated cells.

Finally, the easy handling of these cells make them really useful for crosstalk experiments in co-culture systems (with primary macrophages from bone marrow for example) using trans-wells for example.

Regarding the Rossette step protocol I have not used it so far, since the Magnetic Beads protocol works quite well for us.

Hope this helps,

Hector

Thank you Hector.

One last question about MACS. Do not you have rest of metallic beads? Or after eliminating them... rest of the bound antibodies? The point is... are your cells completely "clean" after isolation?

Hey Sergio,

I haven't used the StemCell Kit, but my concern would be the purity. It already says that it's an "enrichment" kit, so I guess depending on the donor, the purity my vary to some extent (due to the negative selection procedure).

If you would like to use a positive selection method for whole blood, I can recommend the Fab-TACS approach (Kit: https://www.iba-lifesciences.com/cd4-fab-tacs-agarose-column-starter-kit-human/6-3202-002). It is a positive selection approach, but it works with low affinity Fab-fragments instead of high affinity antibodies. You can have a look here for a detailed explanation of the technology:

https://www.iba-lifesciences.com/strep-tag/cell-isolation/#nav-tacs-traceless-affinity-cell-selection

All labeling reagents will dissociate from the cell surface and you can just wash them away, so you don't have any negative effects on downstream applications.

Here you can also have a look at the isolation procedure: https://www.youtube.com/watch?v=0PL_-uNjFZQ

Let me know if you need further explanations :)

Hi Sergio,

I am a Product Manager at STEMCELL Technologies. I checked in with my colleague Coral and she provided the following information on the RosetteSepTM CD4+ Enrichment Cocktail.

The RosetteSepTM CD4+ T Cell Enrichment Cocktail contains Tetrameric Antibody Complexes that binds red blood cells to unwanted cells, forming rosettes. After incubation with the enrichment cocktail, the blood or bone marrow sample is mixed with a density gradient medium (e.g. LymphoprepTM).

The mixture is then centrifuged and the rosettes are pulled to the bottom of the tube due to their increased density. Thus, both red blood cells and unwanted cells are removed in a single centrifugation step, providing enriched CD4+ T cells in a single centrifugation step. When used with SepMateTM, enriched cells can simply be poured off and are ready for downstream applications.

Unlike immunomagnetic cell separation kits (EasySepTM or MACS), with RosetteSepTM no paramagnetic particles are added to your sample and no magnet is required. As well, unlike MACS, no columns are required.

There are a number of different assays that you can run following CD4+ T cell isolation but which one you choose depends on the question you are trying to answer. I hope this helps! If you'd like more help or want to discuss further, please reach out to our Product and Scientific Support team at [email protected]. We will be happy to provide more recommendations once we know more about your research question.

Kind regards,

Danay