40 μm disposable plastic strainer

The immune infiltrate present into the bladder was analyzed by flow cytometry as previously described.^{5 (link)} Briefly, bladders were minced using a scalpel followed by digestion with RPMI medium supplemented with 5% FBS containing 0.5 mg/mL collagenase II (Sigma, Spain) and 1 U/mL DNAse I at 37°C for two-three successive 30 min cycles, with continuous shaking. The cell suspension obtained was filtered through a 40-μm disposable plastic strainer (Becton & Dickinson) and pelleted for staining. The cells were labeled with the following antibodies: PerCP-CD45 APC-Cy7-CD3, FITC-CD4, Alexa 700-CD8, APC-CD62L and BV786-CD127, PE-Dazzle-CD44 and FITC-CD45R/B220 (Biolegend). The lymphocyte gate was defined by morphological parameters, and dead cells were excluded using a live/dead fixable Aqua Dead Cell Stain kit (Invitrogen). Samples were acquired in a Fortessa flow cytometer (Becton & Dickinson), and the data were analyzed using FlowJo software (9.8 v; TreeStar, Portland, OR, USA). Absolute cell numbers were obtained by using Perfect-Count Microspheres (Cytognos). Gating strategy is shown in Supplementary Figure 3.