Hi Maria,

for the FoxP3 staining you can follow the instruction from eBioscience http://tools.thermofisher.com/content/sfs/manuals/staining-intracellular-antigens-for-flow-cytometry.pdf.

For the IL-10 you need to stimulate your T cells with PMA and Ionomycin, blocking the transport outside the cells with BRefeldin or Monensin, and then proceed with the extracellular staining, fix your cells and then stain for Foxp3 and IL10.

This is my protocol for stimulation:

Plate 5x106 splenocytes in flat 24-wells plate. (1ml RPMI 10%FCS)

Final Volume 2 ml RMPI 10%FCS

Add PMA and IONO in 1 ml ( conc 2X)

PMA 20 ng/ml 

IONO 750 ng/ml 

After 1h at 37C, add 2 uL of Monensin/ well (1000x)

Leave at 37C for 4h.

Wash cells and transfer in 96w RB.

e.c. staining in FACS Buffer ( 50 ul/well) 25’, ice, dark

Centrifuge 1500 rpmi, 4 C, 5’.

if you need to use streptavidine, SA 18’, ice, dark.

BD Cytofix/Cytoperm or eBIOSCIENCE 100 ul/well. 20’, ice, dark.

Wash 2 times with BD or eBIOSCIENCE Perm/Wash 1x solution ( diluited 10x in ddH2o).

i.c. staining 50ul/well in PermWASH diluited fluorocrome. 30’,ice, dark.

Hi Caterina,

Many thanks for your answer! I am trying to detect Tregs in peripheral blood and I was wondering whether it would be possible to detect  IL10 also without previous stimulation of cells (naturally IL10-producing cells in periferal blood).