Anti foxp3 apc monoclonal antibody

Draining lymph nodes and spleen cells were harvested and stained for surface markers with anti-CD4-FITC or, separately, anti-CD8-FITC, anti-CD44-V450, and anti-CD62L-APC antibodies (BD Biosciences). To determine intracellular Foxp3 expression, cells were fixed and permeated by Foxp3/Transcription Factor Fixation/Permeabilization kits (eBioscience), and then stained for intracellular FoxP3 with anti-Foxp3-APC monoclonal antibody (eBioscience). The frequencies of CD4⁺Foxp3⁺ Tregs and effector CD8⁺ T cells were finally analyzed through FACSCalibur (BD Biosciences).

In vitro assay to evaluate the inhibition of the transcription factor FoxP3 was performed by flow cytometry. Briefly, CD4⁺ T cells were incubated in the presence of Inducible regulatory T cell (ITR) cocktail, adding indicated EV preparations. The CD4⁺ T cells were seeded at 5 × 10⁴ cells/well in 96 wells plate and pre-activated with anti-CD3 (Tonbo Biosciences clone 145-2C11) and anti-CD28 (Tonbo Biosciences clone 37.51) 1ug/ml, and after 24 h, it was added ITR induction cocktail (100 U/ml IL2, 1 ng/ml TGF-β and 0.1 ul/ml of retinoic acid). The ITRs were harvested, fixed, permeabilized (FoxP3 Tonbo Biosciences Transcription Factor Dye Kit), labeled with anti-FoxP3-APC monoclonal antibody (eBioscience) and FoxP3 expression was evaluated by flow cytometry. The data were processed in FCS Express 5 Flow Cytometry software.