Cd3 biotin

Th17, non-classic and classic Th1 clones were resuspended in complete medium (RPMI 1640) (2 × 10 5 /mL) and seeded on polylysine-coated glass slides. Cells were then incubated for 40 min at 37 °C and 5% CO 2 , followed by 30 min incubation with biotin-CD3 (eBiosciences) or control isotype. Following, cells were washed in PBS pH 7.2 and incubated for 30 min with Streptavidin 488 conjugated (1:200) . Cells were then washed, fixed in formaldehyde (4% in PBS pH 7.2), permeabilized with 0.1% Triton (Sigma) for 10 min, and incubated for 20 min at room temperature with goat serum (1 mg/mL). Cells were then incubated with an anti-Eomes (Abcam 23345, 1:100) or control isotype overnight at 4 °C. Following incubation with ab23345, cells were washed and then incubated at room temperature with anti-rabbit IgG Alexa Fluor 546 conjugated Ab for 30 min (2 μg/mL), in buffer containing TOPRO-3 dye (0.2 μM) for the nuclear counter staining. Cells were then washed in PBS for 5 min, and the slides mounted with Vectashield mounting medium (Vector Laboratories Inc., Burlingame CA). Microscopic images were taken by a LSM 510 META Zeiss confocal microscope system (Carl Zeiss Inc., Jena, Germany), using 40X oil immersion lens, corresponding to a 400X magnification. For image analysis Confocor 2 (Zeiss) software was used.