Dl isocitric acid trisodium salt hydrate

We used a previously established protocol (33 (link)) to treat cord blood-derived HSPCs with approximately IC50 concentrations of treosulfan and thiotepa (4uM and 12,5uM, respectively). Thiotepa treatment was combined with liver enzymes to support conversion to the active metabolites (6 (link)). Used concentrations for these additional compounds were: 0,25% S9 fraction (Aroclor-1254-induced male Sprague Dawley rat liver), 3mM NADP (Sigma) and 15mM DL-isocitric acid trisodium salt hydrate (Sigma).

Extracellular Pel activity was measured by spectrometry as previously described (Matsumoto et al. 2003 (link)). In brief, bacterial cells were cultured in LB broth supplemented with 0.1% (wt/vol) polygalacturonic acid (PGA) at 28°C for 16 h. When needed, 0.4% (wt/vol) sodium citrate, sodium pyruvate, sodium acetate, sodium fumarate dibasic, α-ketoglutaric acid disodium salt dihydrate, or DL-isocitric acid trisodium salt hydrate (Sigma-Aldrich, St. Louis, MO, U.S.A.) were applied. The OD₆₀₀ of bacterial cultures was measured and normalized. Culture supernatant was collected by centrifugation of 1 ml of bacterial cultures at 13,000 × g for 2 min. The supernatant (10 μl) was added into 990 μl of the reaction buffer (0.05% [wt/vol] PGA, 0.1 M Tris-HCl [pH 8.5], and 0.1 mM CaCl₂, prewarmed to 30°C). Pel activity was monitored at an absorbance at 230 nm for 3 min and calculated based on one unit of Pel activity being equal to an increase of 1 × 10 ⁻³ OD₂₃₀ in 1 min.