The effect of Thio-TEPA on slice viability is studied by the determination of intracellular potassium (K⁺) leakage. Intracellular K⁺ is measured by flame photometry and is expressed in relation to the DNA content as measured using fluorometry. Thio-TEPA is added at concentrations of 1, 2, 5, and 10 mM and slices are incubated for 6, 12, and 24 h. The results are compared with the values found for drug-free controls. Thio-TEPA in medium and slices is measured following 3, 6, and 9 h incubation at the three highest concentrations. Krebs-HEPES buffer is used as the slicing buffer and Waymouth's MB 752/1 medium supplemented with 10 μg gentamycin is used as the incubation medium^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Male C57BL/6JIco mice, 19 weeks old, 25 to 30 g, are used as recipients. Congenic C57BL/6J-Gpi-1^a/Gpi-1^a (B6-Gpi-1^a) and BALB.B10LiLa (Gpi-1^a) mice are used as a source of syngeneic and allogeneic donor bone marrow, respectively. The latter combination is H-2-compatible (both H-2^b), but mismatched on a number of minor histocompatibility loci. Thio-TEPA is dissolved in PBS and injected i.p. at a single bolus of 20 mg/kg. Preliminary pilot experiments have established that this approximated to the maximal tolerated dose when higher doses result in lethal gastrointestinal toxicity. Cyclophosphamide (CY) is dissolved in PBS and administered i.p. at a single dose of 200 mg/kg. Total body irradiation is delivered using a ¹³⁷Cs γ-irradiation unit at a dose rate of 87 cGy/min to a dose of 5 Gy. After Thio-TEPA treatment the mice are maintained on neomycin (3.5 g/L sterile drinking water) for 2 weeks to minimize the possible problems of endotoxemia via gut toxicity^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Hagen B, et al. Metabolism and alkylating activity of thio-TEPA in rat liver slice incubation. Cancer Chemother Pharmacol. 1991;28(6):441-7.  [Content Brief]

  [2]. Down JD, et al. Thiotepa improves allogeneic bone marrow engraftment without enhancing stem cell depletion in irradiated mice. Bone Marrow Transplant. 1998 Feb;21(4):327-30.  [Content Brief]