Hello,
I am having inconsistent results from the suppression assay.
The brief protocol:
1. With CD4 microbeads, sorted CD4
--> ~98% of purity was checked each time with flow cytometer.
2. With CD25 microbeads, sorted CD25+ and CD25-.
--> ~2% FOXP3 with CD25- and ~70% FOXP3 with CD25+ were confirmed each time with intracellular staining (ICC and Flow Cytometer).
3. Prior to the co-culture, CD4+ CD25- was stained with Celltrace far red (followed vendor protocol).
4. Co-culture 2.5x104 Celltrace-stained CD25- and dose-dependent CD25+ or CD25- (Tregs:Teff=2:1, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 0:1, 0:2) with CD3/CD28 dynabeads (Cell:Beads=1:0.5, 1:1, 1:1.5, 1:2) in 100ul of RPMI + 1x Glutamax + 50uM beta-mercaptoethanol + 1mM sodium pyruvate + 0.1mM MEM Non-Essential Amino Acids solution + 10mM HEPES Buffer Solution + 1x PEN/STREP + 10% Heat inactivated FBS (with 20IU/mL IL-2 or without IL-2). As the Treg treatment, Naive CD25- and Naive CD25+ were used.
5. Incubated 37C for 4days
The result: Only 1:1.5 cell:beads ratio showed suppression (both with IL-2 and without IL-2). Other three did not show the suppression (both with IL-2 and without IL-2). 1:2 cell:beads ratio showed more suppression in the 0:2 Tregs:Teff ratio well (No matter CD25- and CD25+ as Treg treatment).
I do not know what causes the problem. I heard APC will be better. If the stimulus is the problem, why does APC not cause a problem, but only beads do?
Also, I tried activated CD25- T cells as Teff.
The brief protocol:
1. Sorting the cells by CD4 and CD25 microbeads
2. Activating with CD3/CD28 dynabeads for 1 day or 4day.
3. Removed the beads on day 1 or 4.
3. Prior to the co-culture, CD4+ CD25- was stained with Celltrace far red (followed vendor protocol).
4. Co-culture 2.5x104 Celltrace-stained activated CD25- (1day or 4day activated) and dose-dependent activated CD25+ or CD25- (Tregs:Teff=2:1, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 0:1, 0:2) without activation beads in 100ul of RPMI + 1x Glutamax + 50uM beta-mercaptoethanol + 1mM sodium pyruvate + 0.1mM MEM Non-Essential Amino Acids solution + 10mM HEPES Buffer Solution + 1x PEN/STREP + 10% Heat inactivated FBS (with 100IU/mL IL-2 or without IL-2). As the Treg treatment, activated CD25- (1day or 4 day) and activated CD25+ (1day or 4day).
5. Incubated 37C for 4days.
The result: Without IL-2, all cells are dead. Only IL-2 wells maintain the cells. The suppression is really random. I can't provide specific data, but it seems that FOXP3 does not do anything. The total number of cells looks critical for suppression in the stage of proliferation.
Has anyone done a suppression assay with activated Teff before? If so, can you give advice?
Thanks!
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