Dnasei

For experiments performed on TAMs in vitro, 3 × 10⁵ TC-1/A9 or 1 × 10⁶ TC-1/dB2m cells were inoculated s.c. into mice. Non-necrotic TC-1/A9- and TC-1/dB2m-induced tumors were excised on days 13–14 or 35–40, respectively. The harvested tumors were digested with 1 mg/mL collagenase NB 8 (SERVA, Heidelberg, Germany, 17456) and 100 μg/mL DNase I (SERVA, 8535) in Roswell Park Memorial Institute (RPMI) 1640 medium (without FBS; Sigma-Aldrich, Merck, KGaK, R8758) at 37 °C using the gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), and the cell suspension was filtered through a 70 μm mesh (Miltenyi Biotec). After the removal of erythrocytes with ACK buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.5 M EDTA, pH 7.2–7.4), F4/80⁺ cells were enriched using anti-F4/80⁺ antibody-conjugated magnetic beads (Miltenyi Biotec, 130-110-443) and the autoMACS Pro Separator (Miltenyi Biotec), according to the manufacturer’s instructions. The collected F4/80⁺ cells were used for further experiments. The cells were cultured in DMEM F12 (Biosera, Nuaille, France, LM-D1222) with 10% FBS and antibiotics, as described above (DMEM F12/10).

One million Krebs-2 cells were incubated for 1 h with pUC19 plasmid DNA (0.01; 0.1; 1; 5; 10; 20 μg). Next, to eliminate non-internalized DNA, the cells were treated with DNaseI (#18525, Serva) (10 μg/ml, 37 °С, 1 h), washed once with RPMI-1640 medium (#1.3.4, Biolot, St. Petersburg, Russia), and resuspended in 50 mM EDTA. SDS was added to a final concentration of 1 % and proteinase K (BIO-405010, Bioron GmbH, Germany) treatment (100 μg/ml) was performed at 58 °С for 1 h. Cell lysate was subjected to phenol-chloroform extraction, and the DNA was re-precipitated with 0.6 volumes of isopropanol, washed with 70 % ethanol and dissolved in water (15–40 μl). The DNA thus obtained was transformed into chemically competent XL1Blue MRF' E. coli cells. The cells were spread on agar-Amp plates. Colonies were counted, and this information was used to estimate plasmid copy number per cell. To verify that the transformed cells indeed carried the intended pUC19 plasmid, several individual colonies were grown in LB-Amp overnight. Plasmid DNA was purified and its identity was confirmed by gel electrophoresis.