High five insect cells

Ku70/80 heterodimer, DNA-PKcs, and PAXX were the same purified batches as used in a previous study and documented to be functional^{9 (link)}. L4 with an N-terminal snap-tag and a C-terminal 9Xhis-tag was co-expressed with X4 in High Five insect cells (Invitrogen, B855-02). After thawing on ice, the cell pellets infected with X4 and L4 baculovirus were lysed with buffer of 50 mM sodium phosphate, 500 mM KCl, 0.1% Triton X-100, 20 mM imidazole, 2 mM β-ME, 2 µg/ml aprotinin, 1 µg/ml pepstatin A, 10 µM leupeptin, 1 µM bestatin, and 0.2 mM PMSF, pH 7.8. Then the cell lysate was sequentially passed through Ni-NTA affinity chromatography, anion exchange chromatography and cation exchange chromatography. After cation exchange Mono S column, fractions containing X4L4 were pooled, aliquoted, frozen down using liquid nitrogen, and stored at −80 °C. XLF was expressed and purified using the method reported previously^{37 (link)}. Briefly, XLF cDNA on a pcDNA6 plasmid (pcDNA6/huXLF-Myc-His) was transiently transfected into 293 T cells using the PEI method. The cells were then harvested 48 h after transfection. Ni-NTA chromatography and anion exchange chromatography were then used for XLF purification. The protein concentration was measured by SDS-PAGE gel using BSA as standard.

High Five insect cells (Invitrogen) were routinely sub-cultured to .3–.5×10⁶ cell mL⁻¹ every 2–3 days when cell concentration reached 2–3×10⁶ cell mL⁻¹ in serum-free Insect-XPRESS™ medium (Sartorius) using 125–500 mL shake flasks with a 10% working volume, and maintained at 27°C in a Inova 44R shaking incubator (orbital motion diameter of 2.54 cm—Eppendorf) operating at 100 RPM.