Collagenase type 3

Lung specimens of the young, middle-aged, and old groups were compared. Dewaxed paraffin sections were soaked in distilled water and submerged into a PBS solution containing 0.5-mM calcium chloride (pH 7.4) and 250 units/mL type 3 collagenase (Worthington, Lakewood, NJ, USA) at 37°C for 1.5 h or 3 h [6 (link)]. The collagenase used in the present study was chosen for its substrate specificity to collagens with lower proteolytic activity compared with other collagenases. Digested sections were washed with distilled water before the SAM observation. The same sections were measured at 1.5 h and 3 h after digestion.

Fresh mammary glands from the abdominal/inguinal regions of 6-week-old and 12-week-old female mammary cancer susceptible Wistar-Furth rats were individually collected, scissor minced and digested for 2 h at 37°C in 10 ml of GIBCO Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; ThermoFisher) containing 0.005 g/ml of type 3 collagenase (Worthington-Biochem). Centrifugation was used to remove fat and collect the cell pellets. Individual cell pellets were washed and resuspended in DMEM/F12 media. Each cell suspension was filtered using 40 μm nylon to enrich the mammary ductal fragments and remove stromal cells. The filter was inverted and rinsed to collect the fragments, and the resulting cell pellet containing mammary epithelial cells (MECs) was diluted in PBS and treated for 10 min with 1.5% formaldehyde for DNA/chromatin fixation. After a series of washes, the final cell pellets were collected using centrifugation and stored at −80°C. A total of 6 samples were sent to Arima Genomic, Inc. (n = 3 for 6-week-old and n = 3 for 12-week-old) for Hi-C analysis, consisting of complete sample processing for Hi-C and library preparation and Illumina Next-Generation sequencing.

Single-cell suspension from the spleen was prepared either by pushing the spleen through a 70μm cell strainer, or by enzymatic digest in RPMI supplemented with 2% heat-inactivated foetal calf serum (FCS), 1mg/ml Type 3 collagenase (Worthington) and 0.14mg/ml DNase I (Roche) for 30min with agitation to preserve myeloid cells, which are more fragile and prone to cell death by mechanical dissociation. EDTA was added at a final concentration of 10mM in the final 10min of digest. At the end of dissociation or digest, aggregates were removed by passing through a 100μm nylon filter, then the cell suspension was underlaid with 1ml FCS and centrifuged at 400×g for 7min. Red blood cells were lysed in TAC buffer containing 17mM Tris and 140mM ammonium chloride at pH7.2. Cells were washed twice in FACS buffer (PBS with 2% FCS) containing 5mM EDTA before use in ex vivo assays or for staining with antibodies.

LPMCs were isolated from the SI specimens using a modification of a previously described protocol21 (link)59 . Briefly, the dissected mucosal tissue was incubated in calcium and magnesium-free Hank’s balanced salt solution (HBSS) (Life technologies, Carlsbad, CA) containing 2.5% heat-inactivated fetal bovine serum (FBS) (Life technologies) and 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus. The mucosa was then incubated with agitation in HBSS containing 2 mM and 1 mM EDTA (Quality Biological, Gaithersburg, MD) for 15 min and 45 min, respectively, at 37 °C. The tissues were then collected and incubated with agitation in HBSS containing 400 U ml⁻¹ of type 3 collagenase and 0.01 mg ml⁻¹ of DNase I (Worthington Biochemical, Lakewood, NJ) for 90 min at 37 °C. The insoluble fraction was pelleted, re-suspended in a 40% Percoll solution (GE Healthcare Life Sciences, Pittsburgh, PA), layered on top of a 75% Percoll solution and centrifuged at 2,000 r.p.m. for 20 min at room temperature. Viable LPMCs were recovered from the discontinuous gradient interface.

RAs from 3 young cadavers (40-, 44-, and 45-year-old men; “young RAs”) and 3 old cadavers (82- and 90-year-old men and an 81-year-old woman; “old RAs”) were selected for comparison. Paraffin sections were dewaxed with xylene, soaked in distilled water, and submerged into a solution of phosphate-buffered saline containing 0.5 mM of calcium chloride (pH 7.4) and 250 units/mL type III collagenase (Worthington, Lakewood, NJ, USA) at 37°C for 1.5 h or for 3h [16 (link)]. The collagenase used has substrate specificity to collagens with lower proteolytic activity than other collagenases. Digested sections were first washed with distilled water before being observed with SAM. The same sections were measured 1.5 h and 3 h after digestion.