After washing the coverslips twice in FACS buffer, they were applied onto slides and left to dry overnight. Fluorescence selleck was imaged with Leica TCS SP confocal microscope equipped with an Argon/HeNe laser for double fluorescence at 488 and 633 nm. Confocal images were recorded with a 100× objective and processed with Leica Confocal software. Higher magnification images were composed digitally.

Alexa 488 and TO-PRO-3 iodide were pseudocolored in green and red, respectively. Gain and offset settings were identical for the three-sorted slides. Suppression assays were performed to ascertain the functional ability of the identified Tregs. Isolated mononuclear cells were divided by magnetic separation (MACS) into CD3 positive and negative populations (>90% purity). Per well 25.000 Ibrutinib supplier irradiated (3500 Rad) CD3 negative cells were used as antigen-presenting cells (APC). CD3 positive cells were sorted on a FACS Aria (BD bioscience) according to expression of CD4, CD25 and CD127 in to effector (Teff) and Treg populations (Supporting Information Fig. 1B). Teff cells were identified by positive expression of CD4 and negative expression

of CD25. Tregs were sorted by positive expression of CD4 and CD25 and low expression of CD127. Cells were incubated for 96 h in 37°C, 5% CO2 and stimulated with platebound anti-CD3 (OKT03, 1 g/mL, eBioscience). For the final 16 h, tritium thymidine (3H) was added. The proliferation of Teffs Silibinin and Tregs alone was determined by 3H incorporation. Suppressive capacity of Tregs was assessed in co-culture conditions with equal amounts of Teffs and Tregs. The subsequent proliferation of Teffs in the presence of Tregs was related to the proliferation of Teffs alone. An average value from triplicate wells per condition was set off against medium value.

To further substantiate the functionality of Tregs before and after surgery, CFSE dilution assay was performed on three patients using different ratio of Tregs to PBMCs. 5×104 PBMCs from before surgery were labeled with CFSE according to protocol 49. Cells were cultured as described before with platebound anti-CD3 with different ratios of sorted Tregs from both before and 24 h after surgery. Division of PBMCs was determined after 96 h by analyzing CFSE dilution by means of FACS analysis. To evaluate the role of soluble factors present during the inflammatory response on Treg functionality, a standardized suppression assay was performed in the presence of patient plasma. Teffs (10 000 cells) and Tregs (10 000 cells) were sorted from healthy subjects and co-cultured with 20% heat-inactivated AB serum (Sanquin Blood Bank, Utrecht, The Netherlands) and 20% plasma obtained from patients 4 and 24 h after surgery. Cells were incubated for 96 h in 37°C, 5% CO2 and stimulated with platebound anti-CD3 (OKT03, 1 g/mL, eBioscience). For the final 16 h, tritium thymidine (3H) was added.