Briefly, the mice were anesthetized by pentobarbital sodium injections and perfused via the portal vein with successive pronase E (0.4 mg/ml, Sigma, USA) and collagenase-IV (0.5 mg/ml, Roche, Germany) solutions. The liver tissue was isolated and digested with collagenase IV (1 mg/ml) and DNase (0.02 mg/ml, Roche, Germany) in vitro. The tissue was then filtered through a 100 μm mesh. Cells were separated using Nycodenz gradient (Accurate Chemical, USA) centrifugation. Primary HSCs were seeded in DMEM with 10% FBS, 100 U/ml penicillin sodium, and 100 μg/ml streptomycin sulfate and incubated at 37 °C in CO2.
Collagenase 4
Manufactured by Roche
Sourced in Switzerland, Germany, United States
Collagenase IV is a laboratory reagent that is used to break down the collagen matrix in tissue samples. It is an enzyme that specifically targets and cleaves collagen, a structural protein found in the extracellular matrix of various tissues. Collagenase IV can be used to facilitate the isolation and extraction of cells from tissue samples for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (26 mentions)
Dnase 1, by Merck Group (19 mentions)
Percoll, by Thermo Fisher Scientific (9 mentions)
Percoll gradient, by Merck Group (6 mentions)
Dnase, by Merck Group (5 mentions)
Dispase 2, by Roche (4 mentions)
Collagenase d, by Roche (4 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (4 mentions)
Percoll, by GE Healthcare (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Matrigel, by BD (3 mentions)
Red blood cell lysis solution, by BioLegend (3 mentions)
Red cell lysis buffer, by Merck Group (3 mentions)
Percoll density gradient, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Merck Group (3 mentions)
Pronase e, by Merck Group (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (2 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Canto 1 flow cytometer, by Thermo Fisher Scientific (2 mentions)
90 protocols using collagenase 4
1
Isolation and Culture of Primary Hepatic Stellate Cells
2
Tissue Digestion for Single-Cell Suspension
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The draining inguinal lymph node was collected and chopped into small pieces, then digested in 1 ml RPMI containing 1 mg ml−1 collagenase IV (Worthington Biochemical), 40 μg ml−1 DNase I (Roche, 04716728001) and 2% heat-inactivated FCS for 15 min at 37 °C using a thermoblock. Skin tissue was digested in RPMI containing 1 mg ml−1 collagenase IV, 2 mg ml−1 Dispase II (Roche), 40 μg ml−1 DNase I and 2% heat-inactivated FCS for 30 min at 37 °C. Chopped tumour tissue was digested using 1 mg ml−1 collagenase IV, 40 μg ml−1 DNase I and 2% heat-inactivated FBS for 30 min at 37 °C, and the remaining tumour went through 30 min further digestion using 1 mg ml−1 collagenase D (Roche). Cells were rinsed through a 70 μm cell strainer to obtain single-cell suspensions.
3
Quantifying Neutrophils and Tregs in Tongue
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For quantification of neutrophils, tongues were removed and digested with DNase I (2.4 mg/ml, Roche), and Collagenase IV (4.8 mg/ml) in PBS for 45 min at 37°C. For analysis of Tregs, mice were anesthetized with a sublethal dose of Ketamin (100 mg/kg), Xylazin (20 mg/kg), and Acepromazin (2.9 mg/kg) and perfused by injection of PBS into the right heart ventricle prior to removing the tongue. Tongues were cut in half and the underlying muscle tissue was carefully removed using a scalpel. The remaining tongue tissue was cut into small pieces and digested with DNase I (2.4 mg/ml, Roche), Collagenase IV (2.4 mg/ml) and in some case Trypsin (1 mg/ml) in PBS for 45 min at 37°C. Single cell suspensions were obtained by passing the digested tissue through a 70 μm strainer using icecold PBS supplemented with 1% FCS and 2 mM EDTA and then stained for flow cytometry.
4
Isolation of Immune Cells from Tissues
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Spleens were minced into small pieces and digested in 1 mL of RPMI (Thermo Fisher Scientific) with 200 U/mL collagenase IV (Worthington) and 0.2 mg/mL DNAse I (Roche) for 30 min at 37 °C while shaking. After digestion, cells were passed through a 70 µm strainer and washed once with FACS buffer (phosphate-buffered saline (PBS), 1% fetal calf serum (FCS), 2.5 mM EDTA, 0.02% sodium azide). Erythrocytes were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 2 min at room temperature (RT), washed once, and resuspended in FACS buffer for further analysis. Bone marrow from adult mice was isolated from femurs and tibiae by flushing, and bone marrow from mice under 2 weeks of age was isolated by crushing the bones through a 70 μm cell strainer. Erythrocytes were lysed as above and cells were resuspended in FACS buffer for further analysis. Liver was minced into small pieces and digested in 2 mL PBS containing Mg2+ and Ca2+ (Sigma-Aldrich) with 1 mg/mL collagenase IV (Worthington), 60 U/mL DNAse I (Roche), 2.4 mg/mL Dispase II (Roche), and 3% FCS (Sigma-Aldrich) for 30 min at 37 °C while shaking. After digestion, cells were passed through a 100 µm strainer and centrifuged for 3 min at 50 g at 4 °C, to pellet hepatocytes. The supernatant was collected and recentrifuged for 7 min at 320 × g at 4 °C. Pelleted cells were resuspended in FACS buffer for further analysis.
5
Isolation of Intestinal Immune Cells
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Tissue-specific protocols were used to obtain single-cell suspensions. Following the euthanasia by Avertin injection, the small (ileum) and large (cecum and proximal colon) intestines were rapidly removed and placed in ice-cold PBS. The intestinal tissue was opened longitudinally after removal of fat and connective tissues. Fecal content was removed and the tissue was cut into pieces (approximately 1.0 cm) after washed in ice-cold PBS. Intestinal tissues were then incubated in 5 mL of 5 mM ethylenediaminetetraacetic acid (EDTA) in Hank’s Buffered Salt Solution (HBSS, Invitrogen, Carlsbad, CA) for 30 min at 37 °C with slow rotation (100 rpm). The epithelial cell layer was removed and filtered through 70-μm cell strainers. The retrieved intestinal pieces were washed in HBSS and cut into smaller pieces and immersed in 10 mL digestion solution containing 5% FBS (Sigma-Aldrich, St. Louis, MO), collagenase IV (1.75 mg/mL; Roche, Nutley), and DNase I (0.5 mg/mL; Sigma-Aldrich) at 37 °C for 45 min with slow rotation. MCs were gated for CD45 positive (+) followed by FCεR1+ with CD117 (c-Kit+) expression.
6
Isolation and Analysis of Ankle Immune Cells
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Mice were injected with the K/BxN serum on day 0 and day 2, as described above. On day 10, mice were euthanized and the skin removed from the hind paws. Ankle tendons were cut and the foot was detached from the tibia and placed in DMEM containing 20 units/ml of collagenase IV (Roche). The soft tissue was cut with a scalpel and the joints opened by gentle pulling of the foot bones. The paws were incubated for 2 hours at 37°C with periodic trituration. Liberated cells were collected, strained though a 70 μm filter (Fisher), washed with DMEM, counted and subjected to flow cytometry on Canto I flow cytometer using 7-aminoactinomycin D (7-AAD) to identify live cells, and anti-CD45 (eBioscience clone 30-F11), anti-CD11b (eBioscience clone M1/70) and anti-Ly6G (clone 1A8, BD Pharmingen) antibodies.
7
Isolation of Primary Mouse Hepatocytes
8
Skin Cell Isolation and Characterization
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Skin was cut into pieces and incubated in RPMI containing 10% serum, 0.2 mg/mL collagenase IV (Roche, Basel, Switzerland) and 20000 U/mL of DNAse I (Roche, Basel, Switzerland) for 1 hour at 37°C. Cells were then passed through a 19 G syringe and filtered through a 100 μm cell strainer (BD Biosciences, New Jersey) to obtain a homogenous cell suspension, which was stained with the fluorochrome or biotin-conjugated monoclonal antibodies listed in Supplementary Table 2. Multiparameter analyses of cell suspensions were performed on an LSR II (BD Biosciences, San Jose). Data were analyzed with FlowJo software (TreeStar, Oregon).
9
Isolation of Tumor-Infiltrating CD8+ T Cells
10
Tumor Tissue Dissociation and Cell Analysis
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Tumor tissues were collected and digested with 1 mg/ml collagenase IV (Roche) and 100 μg/ml DNase I (Roche) at 37°C for 40 min. Single-cell suspensions of cells were incubated with FcγRII/III blocking antibody (2.4G2) and stained with specific antibodies followed by established protocol. Samples were analyzed or isolated on BD LSR Fortessa or BD Aria III (BD Biosciences). Data were analyzed by FlowJo software (Treestar).
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