Dear Mildred,

The following link contains some precise protocols that may be helpful. And also Dynabeads provided a product which is efficient for CD4+ T Cell activation.

I hope this information will be helpful to you.

Best,

Alaleh

https://www.bdbiosciences.com/us/resources/s/activationimmunecells

You Need to share some more Details:

1. Do you want to stimulate a) PBMC b) CD8+/CD4+ T cells ?

2. Do you want to stimulate Antigen-specific ?

3. If you expand cells (antigen-specific) you Need to wait 7-10 days before you can restimulate

Thanks Alaleh and Julian.

Alaleh, do you have any experience of using either or both protocols? The BD protocol doesn't mention restimulation (and is much shorter) but the Life technologies protocol does. 

Julian, I want to stimulate isolated  CD4 T cells. I am not sure I understand your second question, please can you clarify? Your third question probably explains the difference between both protocols that Alaleh mentioned above, thank you.

I haven't tried any special protocol with the aim of secreted cytokines measurement but I am using Dynabeads anti CD3/CD28. Anti CD3/CD28 with additional 20-30 U/ml IL-2 (more than this amount leads to Treg expansion) are general (NOT antigen-specific) T cell stimulators.

Of course, I haven't found any official references for this but It is also recommended that immobilized anti CD3/CD28 are more efficient than soluble ones for CD4+ T cells activation while soluble anti CD3/CD28 is better for CD8+ T cells activation.

Thanks Alaleh. I think I may have come across some reference to the point you make about soluble and immobilised cd3/cd28. I am specifically looking to isolate and then stimulate T regs from human PBMCs. Is a combination is anti cd3/cd28 and IL2 sufficient? And what sort of IL-2 concentration is usualy required?

thanks

As far as I know a cytokine cocktail including TGF-b plus IL-2, 100-500 U/ml is used for Treg cells expansion but I am not aware of details.

There are also some kits you can check them.

http://www.ebioscience.com/knowledge-center/cell-type/t-regulatory-cell-expansion.htm

http://www.miltenyibiotec.com/en/products-and-services/macs-cell-culture-and-stimulation/stimulation-reagents/stimulation-based-on-macsibead-particles/treg-expansion-kit-human.aspx

I hope these attachment can help you.

Thank you all for your comments. Does any one know if it is always necessary to use anti-CD3/CD28 or can I just stimulate Tregs with IL-2 to look at cytokine expression? 

Hi All, could I check if anyone has done in vitro stimulation of  isolated T regs and what is sort of T reg number that you start with. The protocol that I will be using suggests 1x10e5 per well (96 well plate) and 1x10e6 per well (24 well plate) which I am unlikely going to get from my limited samples.

Thanks in advance.