All animal studies were conducted following the guidelines approved by Institutional Animal Care and Use Committee (IACUC). Mandibles were isolated from euthanized newborn male CD1 mice. Mandibular molar tooth germs were retrieved from the surrounding tissue, rinsed by sterile phosphate‐buffered saline (PBS), and kept in 1.2 U/ml of DispaseII and incubated at 37°C for 40 minutes. After being washed in plain DMEM medium, epithelium and mesenchyme of the tooth germs were separated with fine needles. The Mesenchymal tissues were treated once with 0.25% trypsin (Sigma), 50 U/mL collagenase I and 20 U/mL DNase I for 10 minutes at 37°C; twice with 100 U/mL collagenase I (Worthington) for 10 minutes at 37°C; and once with 0.25% trypsin and 20 U/mL DNase I for 5 minutes at 37°C. The dissociated cells were plated in 6‐well cell culture plates after washed in complete DMEM, and then incubated for 24 hours at 37°C. Adherent cells were used as TGMCs. Aliquots were kept in a liquid nitrogen tank. All TGMCs used in this study were within 5 passages.
Collagenase 1
Manufactured by Worthington
Sourced in United States
Collagenase I is an enzyme used for the dissociation and isolation of cells from a variety of tissues. It is a mixture of enzymes that specifically cleaves the collagen present in the extracellular matrix. The product is suitable for use in cell culture and tissue engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (22 mentions)
Dnase 1, by Worthington (20 mentions)
Collagenase 2, by Worthington (18 mentions)
Collagenase 4, by Worthington (15 mentions)
Dnase 1, by Merck Group (15 mentions)
Dnase 1, by Roche (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Bovine serum albumin (bsa), by Merck Group (9 mentions)
Dmem f12, by Thermo Fisher Scientific (8 mentions)
Elastase, by Worthington (7 mentions)
Dispase, by Roche (6 mentions)
Macs tissue storage solution, by Miltenyi Biotec (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Dispase 2, by Roche (6 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Calcein am, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (4 mentions)
203 protocols using collagenase 1
1
Isolation and Culture of Tooth Germ Mesenchymal Cells
2
Isolation and Purification of Tumor-associated Immune Cells
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Subcutaneous, orthotopic or MMTV-PyMT tumours were excised, cut in small pieces, treated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U ml−1 DNaseI (Worthington) for 30 min at 37 °C, squashed and filtered. Red blood cells were removed using erythrocyte lysis buffer and density gradients (Axis-Shield) were used to remove debris and dead cells.
Tumour-draining LNs were cut, dissociated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U mL−1 DNaseI (Worthington) for 45 min at 37 °C and filtered.
Spleens were flushed with 200 U ml−1 collagenase III (Worthington) and left for 30 min at 37 °C. Afterwards, spleens were filtered and red blood cells were removed using erythrocyte lysis buffer.
To purify DC subpopulations from tumour, spleen or LNs, CD11c+ cells were MACS-enriched (anti-CD11c microbeads; Miltenyi) and sorted using BD FACSAria II (BD Biosciences) according to the gating strategy inFig. 1a , Supplementary Fig. 6A or Supplementary Fig. 8A , respectively.
Bone marrow leukocytes were isolated through flushing of tibia and femur. The obtained cell suspensions were filtered, and red blood cells were removed using erythrocyte lysis buffer. To purify bone marrow monocytes, CD11b+ cells were MACS-enriched (anti-CD11b microbeads; Miltenyi) before sorting.
Tumour-draining LNs were cut, dissociated with 10 U ml−1 collagenase I, 400 U ml−1 collagenase IV and 30 U mL−1 DNaseI (Worthington) for 45 min at 37 °C and filtered.
Spleens were flushed with 200 U ml−1 collagenase III (Worthington) and left for 30 min at 37 °C. Afterwards, spleens were filtered and red blood cells were removed using erythrocyte lysis buffer.
To purify DC subpopulations from tumour, spleen or LNs, CD11c+ cells were MACS-enriched (anti-CD11c microbeads; Miltenyi) and sorted using BD FACSAria II (BD Biosciences) according to the gating strategy in
Bone marrow leukocytes were isolated through flushing of tibia and femur. The obtained cell suspensions were filtered, and red blood cells were removed using erythrocyte lysis buffer. To purify bone marrow monocytes, CD11b+ cells were MACS-enriched (anti-CD11b microbeads; Miltenyi) before sorting.
3
Collagenase I Solution Preparation
4
Hydrogel Fabrication for Cell Culture
5
Isolation and Dissociation of Mouse Mammary Epithelial Cells
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TEB and duct isolation was performed as previously (Morris et al., 2004 (link)) with modifications. Mammary glands were collected from FVB/n mice at 5–6 weeks of age and coarsely chopped in a Petri dish using scalpel blades before incubation in digest mixture (1 mg/mL collagenase 1 [Worthington] in 10 mL of serum-free L15) at 37°C for 25 min. Following this, the tube was vigorously shaken, and the digest mix was split into two 50 mL Falcon tubes. Tubes were made up to 50 mL with serum containing L15. The tubes were then briefly centrifuged, and the pellets from each tube were combined and centrifuged again before placing onto a gridded 35 mm Petri dish (Thermo Scientific) under a dissecting microscope. Using a 10 μL pipette set to 2 μL, TEBs and ducts were gently sucked up the pipette tip and placed into serum containing L15. Within an hour, TEB and ducts were gently trypsinized to obtain single cells and stained for flow cytometry.
6
Isolation of Single Cells from Lesion Tissue
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The lesion tissue was taken from euthanized mice, minced into small pieces, and incubated for 1 hour at 37°C with agitation in 5 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with collagenase-1 (1.5 mg/ml; Worthington), DNase-1 (0.4 mg/ml; Roche), and 0.5 mM CaCl2. Digestion was stopped by adding 10 ml of PBS with 5 mM EDTA and 2% bovine serum albumin (BSA), and cell suspension was passed through a 70-μm cell strainer to obtain single cells.
7
Isolation and Purification of Hepatocytes and Hepatic Nonparenchymal Cells
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To isolate hepatocytes and hepatic nonparenchymal cells (NPCs), livers of MCS or MCD-fed WT mice, TLR7 KO mice, and IFNAR1 KO mice were perfused (1 mL per minute) and digested with collagenase 1 (Worthington Biochemical Corporation, Lakewood, NJ) after cannulation of portal vein. Liver cell suspension was centrifuged at 50 Â g for 3 minutes. After centrifugation, the pellet representing hepatocytes was resuspended, filtered, and washed several times using Dulbecco's modified Eagle's medium (PAA Laboratories, Piscataway, NJ) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific Inc.), 100 nmol/L dexamethasone (Sigma-Aldrich), 1% insulin-transferrinselenium-X supplement (Thermo Fisher Scientific Inc.), 100 IU/mL of penicillin, and 100 mg/mL of streptomycin. Viability of hepatocytes was assessed using trypan blue (Sigma-Aldrich).
After centrifuging digested liver cell suspension at 50 Â g for 3 minutes, the supernatant containing hepatic NPCs was collected, washed with phosphate-buffered saline, and resuspended in 40% Percoll in RPMI 1640 media. The cell suspension was gently overlaid onto 70% Percoll and centrifuged at 750 Â g for 20 minutes with off-brake setting. NPCs were collected from the interface, washed twice with phosphate-buffered saline, and resuspended in RPMI 1640 media.
After centrifuging digested liver cell suspension at 50 Â g for 3 minutes, the supernatant containing hepatic NPCs was collected, washed with phosphate-buffered saline, and resuspended in 40% Percoll in RPMI 1640 media. The cell suspension was gently overlaid onto 70% Percoll and centrifuged at 750 Â g for 20 minutes with off-brake setting. NPCs were collected from the interface, washed twice with phosphate-buffered saline, and resuspended in RPMI 1640 media.
8
Isolation of Primary Mouse Hepatocytes
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Primary mouse hepatocytes were isolated from male mice at 8 to 12 weeks of age as previously described (60 (link)) with minor modifications. Briefly, collagenase perfusion was performed through the portal vein of anesthetized mice with 50 ml of perfusion buffer (Krebs Ringer buffer containing 3.6 mg/ml glucose, 1 M CaCl2, and 5000 U of collagenase I [Worthington]) at 37 °C. Cells were dispersed, and hepatocytes were collected and plated in collagen-coated plates with Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Cells were cultured for 8 h before further analysis.
9
Isolation of Human Fibroblasts from Foreskin
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Human foreskins were obtained after prepucectomy of foreskins and approval of the protocol by the ethics committee of Army Medical University. The granulation tissues were harvested at 7 days after wounding. The isolation protocols of human fibroblasts, granulation tissue cells and neonatal ROSA26mTmG mouse fibroblasts are described previously20 (link). In Brief, skin tissues of 1-2 cm2 pieces with subcutaneous tissue removal were digested over night at 4°C in a digestion medium containing 1mg/mL dispase (Roche). Following stripping the epidermis, the dermis were cut up and incubated in the digestion medium consisting of DMEM with 0.25% collagenase I (Worthington) at 37 ℃for 1 hour with shaking. The digested cells were then passed through a 75-μm cell strainer, centrifuged, and resuspended in DMEM with 10% foetal bovine serum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime).
10
Tumor Dissociation and Single-Cell Isolation
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All samples were collected in Media199 supplemented with 2% (v/v) FBS. Single-cell suspensions of the tumors were obtained by cutting the tumor into small pieces (1 mm3) followed by enzymatic dissociation for 45 minutes at 37 °C with shaking at 120 rpm using Collagenase I, Collagenase II, Collagenase III, Collagenase IV (all at a concentration of 1 mg/ml, Worthington Biochemical Corporation) and Dispase (2 mg/ml, Gibco) in the presence of RNase inhibitors (RNasin (Promega), RNase OUT (Invitrogen)), and DNase I (ThermoFisher). Erythrocytes were subsequently removed by ACK Lysing buffer (Quality Biological) and cells resuspended in Media199 supplemented with 2% (v/v) FBS for further analysis.
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