Liberase dh research grade

The skin of three non-wounded control Dhh-Cre; tdTomato animals and the 7-day-old wounds of their three littermates were collected post mortem and pooled in two groups. Subsequently, the tissue samples were mechanically chopped using a disposable scalpel and further dissociated to a single cell suspension using 0.25 mg ml⁻¹ Liberase DH Research Grade (05401054001, Roche) in RPMI 1640 (42401, Life Technologies) for 60 min at 37 °C with gentle rocking followed by a treatment with 0.55 mg ml⁻¹ Dispase II (17105, Life Technologies) and 0.2 mg ml⁻¹ DNase I (10104159001, Roche) for 20 min at 37 °C. The cell suspension was then passed through a 40 µm cell strainer and sorted for tdTomato positivity using FACSAria III (BD Biosciences) laser. Sorted cells were directly placed into RNA lysis buffer for subsequent RNA isolation using RNeasy Micro Kit (74004, Qiagen). Total RNA obtained from three control and three wounded skin samples (pools from four wounds) was then amplified using Ovation Single Cell RNA-Seq Multiplex System (0342-32-NUG, NuGEN) and subjected to sequencing on Illumina Hiseq 2500 platform at the Functional Genomics Center Zurich (http://www.fgcz.ch/). Differential gene expression analysis was performed using a minimum fold change of 1.5 and a False Discovery Rate inferior to 0.05. Gene ontology network analysis was performed with MetaCore (Thomson Reuters).

Liberase DH Research Grade (05401054001; Roche Applied Science) was resuspended in sterile water to 2.5 mg/ml concentration and stored in single-use 100 µl aliquots at−80°C. Collagenase/Hyaluronidase (07912; StemCell Technologies) was aliquoted into single-use 250 µl aliquots and stored at −80°C. Upon collection, metastatic tumors were placed into complete cell culture media supplemented with 1X Gibco Antibiotic-Antimycotic (15240-062; Life Technologies) for transportation. Metastatic tumor fragments were minced into 2-mm cubes using scissors and digested in 50 µg/ml Liberase DH (100 µl) and 0.5X Collagenase/Hyaluronidase (250 µl), diluted in 5 mL of McCoy5A serum free media for 4 h at 37°C with gentle agitation by magnetic stirring bar. No undigested tissue was observed. Digested cells were washed twice with complete cell culture media and transferred into 10% FBS McCoy5A media supplemented with 1X Gibco Antibiotic-Antimycotic and 100 µg/ml Primocin (ant-pm-1; InvivoGen).

Techniques for isolation and DRG cell culturing were similar to those previously described (Dembla et al., 2017 (link)). Briefly, DRGs were harvested from all segments and placed into chilled HBSS (Thermo Fisher Scientific, Karlsruhe, Germany). Isolated ganglia were partially digested for 40 min in 1.8 U/ml liberase DH Research Grade (Roche, Mannheim, Germany) at 37°C and digestion was stopped by adding 1 ml culture medium consisting of DMEM, 10% FBS and 1x penicillin-streptomycin (all from Thermo Fisher Scientific, Karlsruhe, Germany). DRGs were triturated with a 1,000 μl pipette and washed once with culture medium. After centrifugation, the supernatant was discarded, and the cells were suspended in culture medium. Subsequently, one tenth to one twelfth of the cell suspension was plated onto the center of a 35 mm plastic dish pre-coated with laminin (Sigma-Aldrich, Munich, Germany). The cells were left to adhere at 37°C in an incubator in a humidified atmosphere containing 5% CO₂ for 1 h after which 2 ml of culture medium were added. Cells were used for measurements on the following day.