The isolation of primary Sertoli cell was performed as previous description with some modifications [16 (link)]. In brief, the testis of 20-day-old mice were decapsulated and washed with PBS for 3 times. The seminiferous tubules were further digested with 2 mg/ml collagenase IV (Sigma, C5138) in PBS for 15 min at 37 °C, and digested with 1 mg/ml hyaluronidase (Sigma, H3506) and 2 mg/ml collagenase IV and in PBS for 10 min at 37 °C. After that, the tubules were then dispersed through pipetting up and down into small short tubules, and centrifuged at 30×g for 3 min at room temperature. The cells were further digested with a mixture of 2 mg/ml hyaluronidase, 2 mg/ml collagenase IV and 0.5 mg/ml DNase I in 1 mg/ml trypsin (Gibco, 25200072) and incubated in 2 ml Eppendorf tubes for 15 min at 37 °C. The digestion was halted using equal volume of F12-DMEM (HyClone, SH30023.01B) including 15% fetal bovine serum (Gibco, 10270). The products were filtered through a 200-mesh filter (Solarbio, YA0961), and washed twice with PBS centrifuged at 600×g for 3 min at room temperature. The separated cells were resuspended with F12-DMEM (HyClone, SH30023.01B), 10 μg/ml insulin, 2.5 ng/ml EGF, and 5 μg/ml transferrin and cultured in 35 mm plastic dishes (CORNING, 4141) at 34 °C and 5% CO2. After 8 h, the dishes were gently washed with PBS twice to remove the unattached germ cells.
Collagenase 4
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, China, Sao Tome and Principe, Israel, Canada, Macao, Italy, Australia, Japan, Switzerland, Senegal
Collagenase IV is a purified enzyme used to dissociate and isolate cells from various tissue types. It is effective in breaking down collagen, a major structural component of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (260 mentions)
Dnase 1, by Roche (104 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (80 mentions)
Hyaluronidase, by Merck Group (67 mentions)
Dnase, by Merck Group (46 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (44 mentions)
Dnase 1, by Thermo Fisher Scientific (33 mentions)
Percoll, by GE Healthcare (30 mentions)
Dmem f12, by Thermo Fisher Scientific (28 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (27 mentions)
Trypsin, by Merck Group (26 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (26 mentions)
Rpmi 1640, by Thermo Fisher Scientific (24 mentions)
Collagenase 1, by Merck Group (22 mentions)
Collagenase 2, by Merck Group (21 mentions)
Trypsin, by Thermo Fisher Scientific (21 mentions)
Percoll gradient, by GE Healthcare (19 mentions)
Hepes, by Thermo Fisher Scientific (18 mentions)
Trypsin edta, by Thermo Fisher Scientific (18 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (16 mentions)
1 113 protocols using collagenase 4
1
Isolation of Primary Murine Sertoli Cells
2
Isolation and Analysis of Splenic Phagocytes
3
Isolation and Culture of Hepatic Stellate Cells
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Hepatic stellate cells (HSCs) were prepared followed by a protocol as described previously [36 (link)]. Briefly, mice were anesthetized and underwent collagenase-pronase perfusion as described above. For quiescent HSC isolation, livers were harvested from healthy mice and digested in a digestive solution containing collagenase IV (100 U/mL; Sigma), Pronase E (mg/mL; Sigma), and DNase I (100 μg/mL; BioDuly) at 37 °C. For activated HSC isolation, livers were collected from fibrotic mice and digested in a digestive solution with collagenase I (100 U/mL; Sigma), collagenase IV (100 U/mL; Sigma), Pronase E (mg/mL; Sigma), and DNase I (100 μg/mL; BioDuly) at 37 °C. After treatment for 40 min, a stopping solution containing DMEM, 10% FBS, and DNase I was added to inactivate the digestive enzymes. Then, the livers were passed through a 40 μm cell strainer, and cell suspension from the digested livers was purified via 8.2% Nycodenz (Axis-shield, Oslo, Norway) gradient centrifugation at 580 g for 20 min at room temperature and filtered through a 40 μm cell strainer [31 (link)]. The obtained HSCs were cultured in plastic dishes with Iscove’s modified Dulbecco’s medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO2. The purity of HSCs was detected to be >99% following the method based on HSC-specific Vitamin A fluorescence as described [36 (link)].
4
Isolation of Lung and Lymph Node Cells
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Lungs were perfused with chilled PBS and then added to RPMI media (Life Technologies, Thermo Fisher Scientific) containing 10 U/mL DNAse I and Collagenase IV (Sigma-Aldrich). Lungs were then mechanically dissociated using a GentleMACS dissociator (Miltenyi Biotec, Sydney, NSW, Australia), before being incubated for 30 min at 37 °C. After incubation, lungs were further dissociated through a 70 μm nylon cell strainer, washed and then resuspended in supplemented RPMI. Red blood cells were removed from single-cell suspension through the addition of 1 mL ACK lysis buffer (Thermo Fisher Scientific), incubation at room temperature (RT) for 45 s and quenching of the reaction with RPMI with 5% FCS. Cells were then washed again with RPMI and resuspended in FACS buffer prior to antibody staining. LNs were prepared with DNAse I and Collagenase IV digestion (10 U/mL, Sigma-Aldrich) and incubation for 20 min at 37 °C. Following incubation, LNs were dissociated through a 70 μm nylon sieve, washed with RPMI and resuspended in FACS buffer prior to antibody staining. Samples were spiked with a known number of Rainbow Calibration Beads (Becton Dickinson Macquarie Park, NSW, Australia), filtered, then run on an LSRII 5L cytometer (BD Biosciences).
5
Isolation of human GPR125+ spermatogonia
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Testicular tissues from 60 OA patients for at least 7 experiments were washed three times in DMEM-containing antibiotics penicillin and streptomycin. Human GPR125-positive spermatogonia were separated using procedures as previously described [21 (link)]. In brief, seminiferous tubules were isolated from human testis biopsies using 2 mg/ml collagenase IV (Sigma) and 1 µg/µl DNase I (Gibco). Human testicular cells were obtained using a second enzymatic digestion with 4 mg/ml collagenase IV, 2.5 mg/ml hyaluronidase (Sigma), 2 mg/ml trypsin (Sigma), and 1 μg/μl DNase I. For differential plating, cells were seeded into culture plates in DMEM/F-12 (Gibco) supplemented with 10% FBS (Hyclone) and incubated at 34 °C in 5% CO2 for 12 h. After incubation, Sertoli cells attached to the culture plates, whereas male germ cells were remained in suspension and collected by centrifugation at 1000 rpm for 5 min. GPR125-positive spermatogonia were separated by magnetic-activated cell sorting (MACS) using an antibody to GPR125 at a dilution of 1:40 pursuant to the procedures in accordance with MACS instruction (Miltenyi Biotec).
6
Isolation of Adult Mouse Cardiomyocytes
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The ALDH2 KO mice were provided by University of Occupational and Environmental Health (Fukuoka, Japan). The cardiomyocytes were isolated from adult male WT mice and ALDH2 KO mice (all were C57BL/6 background) as described previously (Ackers-Johnson et al., 2016 (link); Pang et al., 2016 (link)). Briefly, mice were sacrificed after anaesthetized with 2% isoflurane, and hearts were rapidly excised and mounted onto a temperature-controlled (37°C) Langendorff system (ADInstruments). The hearts were perfused retrogradely through the aorta with collagenase II (Sigma-Aldrich), collagenase IV (Sigma-Aldrich), and collagenase IV (Sigma-Aldrich) for about 20 min until digestion was apparent. The digested left ventricles were then cut into ~1 mm3 pieces and dissociated by pipetting 2 min. After four sequential rounds of gravity settling using three intermediate calcium reintroduction buffers to gradually restore calcium concentration to physiological levels, the cell pellet which was enriched with myocytes was collected and used for the experiments within 8 h after isolation.
7
Hepatic Leucocyte Isolation Protocol
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For the isolation of hepatic leucocytes, livers were perfused with 1 mg/ml collagenase IV (Sigma) and 0.1 mg/ml DNase I (Sigma) in HBSS (Sigma) and minced finely and fragments incubated at 37°C for 40 min in collagenase IV/DNase I solution. After incubation, the cell suspension was pressed through a 70-μM cell strainer (Becton Dickinson, New Jersey, USA), washed twice in cold FACS buffer (PBS containing 2% heat-inactivated FBS and 0.5 mM EDTA), resuspended in 33% Percoll gradient (Percoll, BIO-STRATEGY PTY LIMITED, Brisbane, Australia) in PBS, and centrifuged at 600g for 15 min with no brake. The supernatant containing hepatocytes was discarded, and the resulting non-parenchymal cell pellet was subjected to red cell lysis using Gey’s solution, washed twice, and resuspended in cold FACS buffer.
8
Tissue Dissociation for Immune Cell Isolation
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Mice were sacrificed by CO2 inhalation, perfused with 25 ml ice-cold PBS, and organs were harvested and processed as follows. Lymphoid organs were digested in 0.4 mg/ml collagenase IV (from Clostridium histolyticum; Sigma-Aldrich) in HBSS, including MgCl2 and CaCl2 (Gibco) supplemented with 10% FBS for 30 min at 37°C and agitated. The lung and the liver tissues were digested using 1 mg/ml collagenase IV. For liver samples, a Percoll gradient was performed (continuous gradient, 27%; GE Healthcare). The colon and small intestine were digested as described before (Burkhard et al., 2014 (link)). The skin and tumor tissue were isolated using 1 mg/ml collagenase IV and 0.2 mg/ml DNase I (Sigma-Aldrich) for 1 h at 37°C and agitating.
9
Isolation of Liver and Tumor-Infiltrating Leukocytes
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To isolate liver- or tumor-infiltrating leukocytes (TIL), a liver perfusion was first performed via the portal vein with 15 mL collagenase IV (Sigma, St. Louis, MO) solution. The harvested tumor or live tissues were cut to small pieces and incubated in mixed enzymes including collagenase IV, hyaluronidase, and DNase IV (Sigma) at room temperature; 1.5 hours later, lower speed centrifugation, RBC lysis, and the gradient centrifugation were used to isolate leukocytes. These cells were maintained in RPMI 1640 medium (Cellgro) supplemented with 100 U/mL penicillin, 2 mmol/L L-glutamine, 10 mmol/L HEPES, 50 μmol/L 2-mercaptoethanol, and 10% FBS.
10
Intracerebral Hemorrhage Mouse Model
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ICH models were successfully established by injecting collagenase IV (Sigma-Aldrich, MO, USA) with 0.075U/ 0.4μL PBS into the right basal ganglia in mice as previously reported, briefly, 0.5 mm anterior, 3.5mm ventral, and 2.2mm lateral to the bregma with a rate of 0.1μl/min, as shown in Supplementary Figure 2B . The needle was slowly removed to avoid reflux 5min later. The burr hole was sealed with bone wax, and the incision was sutured. Sham-operated mice underwent the same procedures without the injection of collagenase IV.
PKH26 red fluorescent cell linker mini kit (PKH26, Sigma) was applied for tracking after transplantation. BM-MSCs were trypsinized and resuspended with 2 μM PKH26 dye at room temperature (RT) for 5 min according to the manufacturer’s instructions. Mice were divided into two groups at 24 h after ICH. For the ICH + BM-MSCs treated group, 2×106 BM-MSCs/ 2μl PBS were stereotactically injected into the ipsilateral lesion area with a rate of 0.1μl/min and placed for another 5 min thereafter. For the ICH + PBS treated group, an equal volume of PBS was administrated to the same position.
PKH26 red fluorescent cell linker mini kit (PKH26, Sigma) was applied for tracking after transplantation. BM-MSCs were trypsinized and resuspended with 2 μM PKH26 dye at room temperature (RT) for 5 min according to the manufacturer’s instructions. Mice were divided into two groups at 24 h after ICH. For the ICH + BM-MSCs treated group, 2×106 BM-MSCs/ 2μl PBS were stereotactically injected into the ipsilateral lesion area with a rate of 0.1μl/min and placed for another 5 min thereafter. For the ICH + PBS treated group, an equal volume of PBS was administrated to the same position.
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