Anti ctla 4 pe efluor610 clone uc10 4b9

Splenocytes cultured in complete medium (RPMI 1640 medium supplemented with 10% FCS, Sodium Pyruvate, L-glutamine, antibiotics, and 2-Mercaptoethanol) were stimulated with PMA (50 ng/ml) and ionomycin (IO) (1 μg/ml) for 4 h at 37°C. The cells were stained as mentioned above, but anti-CD44-APC-efluor780 (clone IM7; eBioscience) and anti-CTLA-4 PE-eFluor610 (clone UC10-4B9; eBioscience) were also included in the surface staining.

Cells from cLN cultured in complete medium (RPMI 1640 medium supplemented with 10% FCS, sodium pyruvate, L-glutamine, antibiotics and β-mercaptoethanol) were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 h at 37°C. The cells were stained as mentioned above, but anti-CD44-APC-efluor780 (clone IM7; eBioscience) and anti-CTLA-4 PE-eFluor610 (clone UC10-4B9; eBioscience) were also included in the surface staining. For the other Treg effector markers, non-stimulated cells from cLN were stained as mentioned above, but anti-CD44-AlexaFluor700 (clone IM7; BD Pharmingen), anti-CD62L-eFluor450 (clone MEL-14; eBioscience) and anti-KLRG1-APC (clone 2F1; eBioscience) or anti-TNFR2-BV421 (clone TR75-89; BD Pharmingen) and anti-ICOS-PE-Cy5 (clone 7E.17G9; eBioscience) were also included in the surface stain.