I am trying to activate T cells by stimulating WBC with TNF-alpha and/or histamine. I hope to detect an activation status of CD3 cells detectable by CD69 over-expression (as a model of inflammatory activation). Do anyone share a protocol to stimulate WBC with TNF-alpha/histamine in order to observe this activation?
You might have a look at this paper
Article Antigen-independent activation of naive and memory resting T...
I agree with Sayed.. PMA and ionomycin will stimulate them efficiently . Please be aware that the surface markers for CD4 will internalise but you should be OK with CD3. You will certainly see CD69 increase on your T-cells indication activation as I have tested this
BD cytofix/cytoperm kit has a good protocol. My modified version is below
1. Label 4 Falcon tubes with patient name and Stim/Unstim.
2. In tubes 1 (marked un-stim) and tube 2 (marked stim) do the following:
3. Place 500µl IMDM (supplemented with Pen/Strep) in Stim (1) / Unstim (2) tubes, add 500 µl whole blood to each and mix well
4. Add 50ng/ml (10 µl) PMA [ 5µl neat PMA in 995µl IMDM] to stimulated cell tube
5. Add 1µM (10 µl) Ionomycin [5µl neat Ionomycin in 495µl IMDM]stimulated cell tube
6. Add 1µl of GolgiPlug protein transport inhibitor to both stimulated and unstimulated tubes.
7. Flick tube to mix. Aliquot 200µl into new 12 x 75mm falcons
Tubes 3 [marked name & unstim], and 4 [marked name &stim].
8. Incubate overnight in 5% CO2 at 37degrees.(18hrs). Retain original tubes 1 &2 as backups.
Hi yes Robert..good idea and why not HLA-DR a great marker of T-cell activation
The most consistent way to achieve long-term activation is using anti-CD3 and anti-CD28. There are commercially available beads that display these antibodies on their surface. You can also coat your own plates. For a few hours activation, you can use PMA/ionomycin. CD4 will shift leftward on flow as mentioned above.
PHA stimulation will have a good effect in stimulating the cells..
Thank you everyone! In particular Piero Pileri to share this interesting paper and Kevin Marron for his protocol. I tried a stimulation with TNF-alpha on different time/dose conditions because of the study was to investigate its effect on T cells. No activation by CD69/HLA-DR was appeared in the T cells. I will procede by PMA/ionomycin (because I have these products on my laboratory at this time). Thank you very much.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology