Macs m tube

Biopsy specimens were thawed on ice before being cut into approximately 30-mg sections. One section was added to a MACs M tube (Miltenyi Biotech, UK) containing 1 ml Qiazol (Qiagen, UK) and dissociated on a GentleMACs instrument (Miltenyi Biotech, UK) using the manufacturer’s RNA settings. The sample was centrifuged and incubated at room temperature (RT) for 5 min before transferring the lysate to a 1.5-ml centrifuge tube. Proteinase K (20 μl) was added to the sample before being incubated at 56 °C for an hour. Chloroform (200 μl) was added, and the mixture was shaken vigorously for 15 s. The sample was then incubated at RT for 2 min before being centrifuged at 12,000 × g for 15 min at 4 °C. The upper aqueous phase was transferred to a fresh 1.5-ml centrifuge tube before the addition of a 1× volume of 70 % ethanol. The sample (up to 700 μl) was added to an RNeasy minispin column (Qiagen, UK) and centrifuged at RT at 8,000 × g for 30 s. Any remaining sample was also passed through the column. All remaining steps were performed according to the manufacturer’s guidelines, with elution in 30 μl of RNase-free molecular-biology-grade water (Thermo Fisher, UK).

The mouse experiments were performed as previously described (Pawar et al., 2015 (link)). Briefly, 7- to 8-week -old female BALB/c mice (Janvier, Germany) were injected intradermally with 5 × 10⁵ cells of the colon carcinoma cell line CT26. Mice bearing tumors of 150–200 mm3 diameter were infected intravenously (i.v.) with 5 × 10⁶ colony forming units (CFU) of the PA14 strains suspended in phosphate-buffered saline (PBS). All animal experiments were carried out with the permission of the lower Saxony authorities (LAVES—permission No. 33.9-42502-04-12/0713). At the indicated times, the mice were sacrificed and the tumors were homogenized in 2 ml of ice-cold 0.1% (v/v) Triton X-100/PBS using gentle MACS™ M-tubes and a dissociator from Miltenyi Biotec. The samples were serially diluted and plated on Luria-Bertani (LB) agar plates containing ampicillin (0.1 mg/ml).