Quantification of Tacrolimus Levels

Blood samples were drawn pre-dose and 1.5, 48, 96, and 192 h after drug administration. The samples for whole blood PK measurement were collected in K2EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, US) and stored at −80 °C. PBMCs were collected using sodium heparin CPT tubes (Becton Dickinson), and T cells were isolated from heparinized whole blood by immunomagnetic cell sorting. The RoboSep human T cell isolation kit was used in combination with RoboSep (StemCell Technologies) to label unwanted cells with antibody complexes and magnetic particles, after which T cells were isolated by automated magnetic sorting. After PBMC and T cell isolation, the cells were washed, and the remaining red blood cells were removed using RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, US). PBMCs and T cells were counted with a MacsQuant 10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and stored in PBS at 20 × 10⁶ cells/mL at −80 °C. The purity of the isolated T cell population was assessed with an anti-CD45-FITC and anti-CD3-VioGreen staining (Miltenyi Biotec).
Whole blood and intracellular tacrolimus concentrations were measured using a Waters Acquity UPLC–MS/MS system by the Department of Hospital Pharmacy, Erasmus Medical Center, as described previously [19 (link)].