Puradisc 25 syringe filter 2 μm

R. typhi (strain Wilmington, accession no. AE017197) was cultured in L929 mouse fibroblasts (ATCC CCL-1) in RPMI1640 (PAA, Cölbe, Germany) supplemented with 10% FCS (PAA, Cölbe, Germany), 2 mM L-glutamine (PAA, Cölbe, Germany) and 10 mM HEPES (PAA, Cölbe, Germany) without antibiotics (standard culture medium). 1×10⁷ γ-irradiated (1966 rad) L929 cells were seeded in 175 cm² culture flasks (Greiner Bio-One, Frickenhausen, Germany). One day later cells were infected with R. typhi and incubated for 5 to 7 days. For the preparation of bacterial stocks, infected L929 cells were resuspended in 1.5 ml PBS. 200 μl silicium particles (60/90 grit silicon carbide; Lortone inc., Mukilteo, USA) were added and cells were vortexed thoroughly for 1 min. The crude lysate was strained through a 2 μm cell strainer (Puradisc 25 syringe filter 2 μm; GE Healthcare Life Sciences, Freiburg, Germany). Bacteria were centrifuged at 4300×g for 5 min at room temperature and frozen in FCS with 7.5% DMSO in liquid nitrogen in Cryo.S tubes (Greiner Bio-One, Frickenhausen, Germany). Thawed bacterial stocks were centrifuged at 6200×g for 5 min at room temperature, washed twice with PBS and analyzed for bacterial content by quantitative real-time PCR (qPCR) and immunofocus assay as described previously [48 (link)] to determine spot forming units (sfu).

The Wilmington strain of R. typhi was used for all experiments. γ-Irradiated (1,966 rad) L929 cells (6 × 10⁶) in 25-cm² cell culture flasks were infected with the bacteria. For the preparation of bacterial stocks, cells were harvested 1 week postinfection. The cells were resuspended in 1.5 ml RPMI 1640 cell culture medium and vortexed thoroughly for 1 min with 200 μl sterile silica particles (60/90 grit silicon carbide; Lortone Inc., Mukilteo, WA, USA). The supernatant was passed through a 2-μm-pore-size filter (Puradisc 25 syringe filter; 2 μm; GE Healthcare Life Sciences, Freiburg, Germany). Bacteria were collected by centrifugation of the flowthrough at 7,826 × g (5 min; 4°C), washed, and resuspended in 250 or 300 mM aqueous sucrose for electroporation. For the preparation of stocks of wild-type R. typhi and transformants, the bacteria were resuspended in cell culture medium supplemented with 50% fetal calf serum (FCS) and 7.5% dimethyl sulfoxide (DMSO) and frozen in liquid nitrogen. For the infection of mice and cell cultures, bacterial stocks were thawed and centrifuged at 7,826 × g. The bacteria were used directly after washing in PBS. The bacterial content of the stocks was determined by qPCR. Spot-forming units (SFU) were determined by immunofocus assay, as previously described (31 (link)).