Asparaginase

Mice were injected intravenously with 3 IU of asparaginase per g of body weight (Takeda). At the indicated times after injection, mice were euthanized by CO 2 /O 2 asphyxiation, a blood sample was drawn, and tissues of interest were dissected. When indicated, mice were pretreated with 150 mL (1 mg/mL clodronate) of liposomes or control liposomes (12) via tail vein injection 24 h before administration of 111 In-labeled asparginase. Residual asparaginase activity in serum was determined by photometric detection of the ammonia release after reaction with Nessler reagent (Sigma-Aldrich). In brief, 15 mL of serum were diluted with 60 mL of 44 mM L-asparagine (Sigma-Aldrich) dissolved in 15 mM Tris-HCl buffer, pH 7.3, supplemented with 0.015% (w/v) bovine serum albumin and incubated at 37°C for 30-45 min. The reaction was stopped by the addition of 50 mL of trichloroacetic acid (24.5% [w/v]; Sigma-Aldrich). After centrifugation, 15 mL of the supernatant was added to 120 mL of Nessler solution diluted with deionized water (1:8). The optical density of the solution was measured at 450 nm using the Multiskan Ascent plate reader (MTX Lab Systems).

Human peripheral blood mononuclear cells (PBMCs) were isolated from blood obtained from healthy volunteers upon informed written consent, using density gradient centrifugation (Lymphoprep; Axis-Shield). Asparaginase (Takeda) was conjugated with fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Pierce / Life Technologies). PBMCs or THP-1 cells were incubated with FITC-labeled Asparaginase in culture medium for 1 h at the indicated temperatures. After incubation, PBMCs were stained for expression of lineage markers with anti-CD14, -CD45, -CD3, and -CD19 antibodies purchased form BD Biosciences and analyzed using a LSRII flow cytometer (BD Biosciences). The data were collected and analyzed by FlowJo software (version 8.8.7; FlowJo).