Collagenase dispase

CAFs were isolated from MMTV+;FSP-Cre−;FAK^fl/fl and MMTV+;FSP-Cre+;FAK^fl/fl mice as described previously^{62 (link)}. Tumours were cut into small pieces and digested for 1 h at 37 °C in 5 mL collagenase/dispase (1 mg/mL; Sigma). After filtering the undigested tissue, the solution was centrifuged (3000 g, 5 min) and the final pellet was resuspended in DMEM high glucose, 10% FBS, 1% ITS and seeded on a 6 cm dish. After 30 min, fibroblasts are adhered and non-adherent tumour cells were replated and cultured as described above. Adherent fibroblasts were used after 1–3 passages.

Mice were anesthetized with 1% of sodium pentobarbital through intraperitoneal injection (70 μL/10 g). Then abdominal aorta was intersected, and mice were perfused through right ventricle with 10 mL of phosphate-buffered saline (PBS) to reduce lung blood content. 3 mL trypsine (%) to initiate the fine digestion, 10 mL PBS to wash out trypsine, 3 mL collagenase-dispase (3 mg mL⁻¹ collagenase (Sigma-Aldrich, St. Louis, MO, USA) 1 mg mL⁻¹ dispase (Roche F. Hoffmann-La Roche Ltd. Basel, Switzerland) 1u μL⁻¹ DNAse I (Sigma-Aldrich). Finally, the lung was filled up with the collagenase-dispase solution trough the trachea. Lungs were removed from the chest and separated from the heart and thymus and cleaned from connective tissue. Pulmonary lobes were dissected into smaller pieces and digested in 10 mL collagenase-dispase for 50 min with continuous stirring. Digested lung cells were filtered with 70-μm cell-strainer (BD Becton, Dickinson and Company Franklin Lakes, NJ, USA)

Mice were anesthetized intraperitoneally with ketamine-xylazine (5 mg/kg), sacrificed by cervical dislocation, and their lungs were excised. Lung tissues were divided into three groups and were stored in RNA-later (Qiagen; Hilden, Germany) for molecular biological studies, in 1 ml lysis buffer for Western blot analysis or in 4% buffered formaldehyde (Szkarabeusz Ltd., Pecs, Hungary) for immunohistochemical examinations.
For the cell sorting procedure, 3 mice/group were anesthetized. Abdominal aorta was intersected, and mice were perfused with 10 ml of PBS through right ventricle to reduce blood content of the lung. To initiate the fine digestion 3 ml trypsin was applied, 10 ml PBS was used to wash out trypsin thereafter. 3 ml collagenase-dispase 3 mg/ml collagenase (Sigma-Aldrich, St. Louis, USA), 1 mg/ml dispase (Roche F. Hoffmann-LaRoche Ltd., Basel, Switzerland), and 1 u/μl DNase I (Sigma-Aldrich, St. Louis, USA) were used to fill up the lungs through the trachea. Pulmonary lobes were dissected into smaller pieces and digested in 10 ml collagenase-dispase for 50 min with continuous stirring. Digested lung cells were filtered with 70 µm cell-strainer (BD Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Primary neuron cultures were established as previously described^{19 (link),32 (link),33 (link),60 (link),73 (link)}. In brief, 20 tribolium or 2 locust brains per culture were dissected and collected in Leibowitz 15 medium (Gibco; Life Technologies, Darmstadt, Germany) supplemented with 1% penicillin/streptomycin and 1% amphotericin B (both Sigma-Aldrich, Munich, Germany) (from now referred to as L15 medium). Subsequently, brains were enzymatically digested in collagenase/dispase (2 mg/ml, Sigma-Aldrich, Munich, Germany) for 45 min (T. castaneum) or 30 min (L. migratoria) at 27 °C. Enzymatic reaction was stopped by repeated washing in Hanks’ balanced salt solution and brains were mechanically dissociated by repeated pipetting in L15. The suspension of dissociated brain cells was seeded on Concanavalin A (Sigma-Aldrich, Munich, Germany) coated coverslips and let to rest for 2 h. Afterwards, culture dishes were filled with L15 supplemented with 5% fetal bovine serum gold (FBSG, PAA Laboratories GmbH, Pasching, Austria). Medium was replaced by L15 plus FBSG on day two and by L15 without serum on day four in vitro. Primary cell cultures were maintained at 27 °C without CO₂ buffering.

Primary spinal cord ECs were isolated, as our previous study described [26 (link)]. In brief, the spinal cord was obtained, then minced until a milky suspension. After centrifuging, the supernatant was removed. Following 0.1% (v/v), collagenase type II (Sigma, USA) was added to digest for 1 h, and the residue was collected after centrifuge. To remove the residue’s myelin, 20 ml of BSA-DMEM (20%, w/v) was added. Following the upper myelin discarded, 0.1% (v/v) collagenase/dispase (Sigma, USA) was used to digest into single cells. After centrifuging and discarding the supernatant, cells were seeded into cell culture plates for the next experiment.

Four 2-week-old IRS-1 transgenic SD rats and control rats were anesthetized with ether and were sacrificed by decapitation. Brain tissues were minced and gently dissociated in buffer containing 15 mM Hepes (pH 7.4), 153 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2x 2H₂O, 2.6 mM MgCl₂x6H2O, and 1% Bovine Serum Albumin (BSA). The resulting microvessel fraction was then sequentially digested with collagenase/dispase at a concentration of 1 mg/mL (Sigma-Aldrich) for 45 min at room temperature. After centrifugation, the pellet containing primary endothelial cells (PEC) was resuspended in Dulbecco’s modification of Eagle’s medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 4 μg/ml puromycin in a humidified incubator (37 °C, 5% CO2). Cells from passage 2 to 5 were used.