Collagenase type 1a

Flow cytometry analysis of tissue samples was performed as previously described (49 (link)). Briefly, mice were euthanized via CO₂ asphyxiation, and the right forelimb was dissected. The implant tube and surrounding tissue were removed, weighed, and digested in collagenase type 1A (1 mg/ml, Sigma) at 37°C for 45 min. Following digestion, samples were separated using a cell strainer to form a single-cell suspension. The single-cell suspensions were stained for analysis using standard methods. The samples were analyzed on a FACSAria III flow cytometer (BD Biosciences). The antibodies used for cell staining were as follows: Alexa Fluor 488–conjugated anti-CD206 (BioLegend), BV421-conjugated anti-CD19 (BioLegend), BV605-conjugated anti-CD4 (BioLegend), BV785 anti-CD8a (BioLegend), phycoerythrin (PE)/Cy7–conjugated anti-CD3ε (BioLegend), BV510-conjugated anti-Ly6C (BioLegend), allophycocyanin (APC)–conjugated anti-F4/80 (BioLegend), APC/Cy7-conjugated anti-Ly6G (BioLegend), and PE-conjugated anti-CD86 (BioLegend). Live/dead staining was performed using the Zombie Red Fixable Viability Kit per the manufacturer’s instructions (BioLegend). Precision counting beads (BioLegend) were used to report absolute cell numbers.

Germ cells of E8.5 to E18.5 embryos were isolated from OG2 (Oct4ΔPE-GFP)/CD1 mice by disaggregating the genital ridges of E8.5 to E11.5 embryos and the fetal gonads of E12.5 to E18.5 embryos. Disaggregation was performed by enzymatic digestion with collagenase type 1A (Sigma) and trypsin-EDTA (0.25%) at a ratio of 1∶2 with shaking at 700 rpm for 8 min at 37°C. Single-cell suspension was achieved more efficiently by triturating the cell aggregates. Enzymatic digestion was stopped by adding a two-fold excess of DMEM/F12+10% FBS. Cell suspensions were washed once in DMEM/F12+10% FBS and FACS sorted for GFP. GFP-positive PGCs were directly sorted into lysis buffer RLT (QIAGEN, Hilden, Germany) using a FACSAria cell sorter (BD Biosciences). Single-cell suspensions of GFP-positive PGCs were analyzed using a FACSAria cell sorter (BD Biosciences). Data analysis was done using FlowJo software (Treestar).

Experimental protocols were approved by the Simon Fraser University Animal Care Committee (Protocol 1040K-08) and were in accordance with and Canadian Council on Animal Care. Oocytes were isolated from female Xenopus laevis frogs that were terminally anesthetised by immersion in 2 g/L tricaine solution (Sigma Aldrich) for 10–15 min. The ovarian lobes were surgically removed and partial digestion of follicular layers was achieved upon treatment with 1 mg/ml collagenase type 1A (Sigma Aldrich) in a calcium free solution (MgOR2) containing (in mM): 96 NaCl, 2 KCl, 20 MgCl₂, 5 HEPES (titrated to pH 7.4) for 1 h. This was followed by manual removal of the remaining follicular layer of selected stage V-VI oocytes. Defolliculated oocytes were then injected with 50 nl (5–15 ng) of cRNA using a Drummond digital micro dispenser (Fisher Scientific, Nepean, Canada). After injection, oocytes were incubated in SOS+ medium containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, 2.5 sodium pyruvate, 100 mg/L gentamycin sulfate and 5% horse serum (titrated to pH 7.4) at 19 °C for 2–5 days prior to electrophysiological recording.

The pathogen-free C57BL/6 mice (male, 5 wks old) were from Laboratory Animal Center of Xiamen University (Xiamen, China). All animal procedures were in strict accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals. All the experimental protocols were approved by the Animal Care and Use Committee of Xiamen University following the Guide for the Care and Use of Laboratory Animals [22 (link)]. The mice had free access to water and were fed with a control diet. Every effort was made to minimize the number of animals used and to minimize suffering. After the mice had been euthanized by pentobarbital administration (150 mg/kg) followed by cervical dislocation, the kidneys were removed immediately and thin coronal sections (1-mm) were cut with a razor blade. The TALs were dissected in a HEPES-buffered NaCl solution containing 1 mg/mL collagenase type 1A (Sigma, St. Louis, MO) at 37°C for 55 minutes. Then, the dissected TAL was transferred onto a cover glass (5 mm × 5 mm) that was coated with poly-lysine (Sigma) overnight to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Olympus) for single channel recordings and the tubules were superfused with HEPES-buffered NaCl solution containing (mM): 5 KCl, 140 NaCl, 1.8 MgCl₂, 1.8 CaCl₂ and 10 HEPES with pH of 7.4.

Lungs were excised, cut into small sized pieces and digested in RPMI medium supplemented with 100 µg/ml DNAse (Sigma-Aldrich) and 1 mg/ml Collagenase Type 1A (Sigma-Aldrich) at 37°C. Digested lungs were filtered through a 70 µm cell strainer, pelleted (400 G, 4°C, 5 min) and resuspended in 6 ml 40% percoll in RPMI (v/v) solution, which was underlayed with 4 ml 80% percoll solution (GE Healthcare-Life Sciences). Tubes were centrifuged (1600 G, RT, 15 min) with brake set to 0. Lymphocytes were collected from the interphase and analyzed by flow cytometry.

After removing testis from the scrotum, decapsulated
tissue was minced mechanically by multiple
aspirations through pipette tips and after
complete disassociation of the tubules they were
transferred into the culture medium Dulbecco’s
Modified Eagle Medium (DMEM/HAMF12; Gibco,
USA). Digestion was conducted in two steps.
In the first step, in order to obtain testicular cell
fraction only collagenase Type 1A (1 mg/ml, Sigma,
Germany) was added to the medium. Digestion
was performed for 10 minutes in a shaking
water bath operated at 110 cycles per minute. The
fraction was separated by sedimentation at unit
gravity. Tubules were allowed to settle by gravity
and washed by phosphate buffered saline (PBS).
Supernatant containing the interstitial cells was removed
and the cell fraction was stored in DMEM/
HAM F12. In the next step, to obtain a fraction
consisting of large proportion of germ cells, sertoli
cells and peritubular cells, the fragments obtained
after the first digestion were washed in DMEM and
digested in a mixture of collagenase type IA (1 mg/
mL, Sigma, Germany), DNase (0.5mg/ml, Sigma,
Germany), and hyaluronidase (0.5 mg/ml, Sigma,
Germany). Digestion was performed at 32˚C for about 10 minutes. After washing, the single-cell
suspension mainly consisted of germ cells, sertoli
cells and also Peritubular cells. The efficiency of
the digestion was evaluated microscopically.