Liberase

Manufactured by Merck Group

Sourced in United States, Germany

Liberase is a proprietary enzyme blend designed for the gentle and effective dissociation of tissues and cells. It is a versatile tool used in a variety of research and clinical applications, such as cell isolation, tissue engineering, and regenerative medicine.

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Liberase TL (117 citations) Liberase DH (18 citations) Liberase TH (17 citations) Liberase TM Research Grade (7 citations) Liberase Blendzyme 3 (4 citations) Liberase enzyme blend (3 citations) Liberase TM solution (2 citations) Liberase/DNAse I (2 citations) Liberase TL Research Grade (2 citations) Liberase protease (2 citations)

268 protocols using liberase

Single-cell Isolation from Lymph Nodes and Skin

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Mice were euthanized by cervical dislocation. Single‐cell suspensions were prepared from the footpad and skin‐draining popliteal lymph nodes. LNs were first incubated in Liberase TM (1.67 Wünsch units/ml) (Sigma) in PBS with 25 mM HEPES for 30 min at 37°C before adding PBS with 25 mM HEPES + 10% FBS to halt the digestion process, followed by mechanical disruption of the tissue with 21 gauge needles (BD) and 1 ml syringes (BD) and then filtered through a 70‐μm mesh. For skin, the footpads of mice were skinned and finely chopped up with surgical scissors in 2 ml safe‐lock tubes (Eppendorf). Processed skin was then incubated in Liberase TM (1.67 Wünsch units/ml) (Sigma) in PBS with 25 mM HEPES for 60 min at 37°C on a rotator before adding PBS with 25 mM HEPES + 10% FBS to halt the digestion process. Samples were then filtered through a 70‐μm filter.

Stotesbury C., Wong E.B., Tang L., Montoya B., Knudson C.J., Melo‐Silva C.R, & Sigal L.J. (2020). Defective early innate immune response to ectromelia virus in the draining lymph nodes of aged mice due to impaired dendritic cell accumulation. Aging Cell, 19(7), e13170.

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Isolation of Mouse Hepatocytes

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Primary cell culture was performed as described previously^{30 (link)}. The hepatocytes of mice were isolated by collagenase digestion and the Percoll Gradient method. In detail, mice were anesthetized and perfused with Ca²⁺ and Mg²⁺ free-Hanks’ Balanced Salt Solution (HBSS) containing EDTA (1 mM), and then digested with a collagenase solution containing liberase (0540119001, Sigma). Livers were removed and rinsed twice with HBSS, digested with a collagenase solution containing liberase (0540119001, Sigma-Aldrich) and then gently teased with forceps until they were in solution.
The cell suspensions were filtered through a sterile 40 μm nylon cell strainer (93040, SPL) to remove undigested tissue and connective tissue. The cells were centrifuged for 5 min at 1000 rpm and resuspended in medium. The resuspended cells were centrifuged using 40% Percoll for 15 min at 2000 rpm with the brake option off. After centrifugation, the healthy hepatocytes were washed twice with DMEM supplemented with 5% FBS, and then seeded into well tissue culture plates.

Lee S.R., Choi W.Y., Heo J.H., Huh J., Kim G., Lee K.P., Kwun H.J., Shin H.J., Baek I.J, & Hong E.J. (2020). Progesterone increases blood glucose via hepatic progesterone receptor membrane component 1 under limited or impaired action of insulin. Scientific Reports, 10, 16316.

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Lung Cell Senescence Assessment

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After euthanasia, mice were immediately perfused with PBS containing 2 mg/ml Liberase (Sigma-Aldrich, St. Louis, MO) via cannulation of the right cardiac ventricle. Whole lung lobes from perfused mice were place in cold Hank’s buffered salt solution (HBSS) and mechanically dissociated using a GentleMACS tissue dissociator and tubes (Miltenyi-Biotec, Bergisch Gladbach, Germany), as previously described [14 (link)]. Samples were then incubated for 30 minutes at 37°C 5% CO₂ with 2 mg/ml Liberase and 0.1 mg/ml DNase I (Sigma-Aldrich, St. Louis, MO). After dissociation, samples were filtered through 70 μM nylon mesh filters, washed with HBSS, and spun down for 5 minutes at 300 × g, 4°C. Samples were then resuspended in 5 mL ACK (ammonium-chloride-potassium) lysis buffer for 5 minutes at room temperature to remove red blood cells. After ACK lysis was quenched with 15 mL cold HBSS, cells were spun for 5 minutes at 300 × g, 4°C and resuspended in PBS + 0.1% BSA for cell counting. Samples were stained for SA-β-gal activity staining by incubation for 30 minutes in staining buffer and colorimetric substrate from the CellEvent Senescence Green Detection kit (Invitrogen, Waltham, MA) in a 37°C non-CO₂ incubator, then washed with PBS and stained for flow cytometry as described below. Fluorescence minus one controls without substrate were used to control for background signal.

Tobin J.F., Laban A, & Wirth D.F. (1991). Homologous recombination in Leishmania enriettii.. Proceedings of the National Academy of Sciences of the United States of America, 88(3), 864-868.

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Isolation of Adult Mouse Ventricular Myocytes

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Adult mouse ventricular myocytes were isolated from the LV of 1.5‐month‐old mice using a retrograde Langendorff perfusion system and enzymatic digestion (0.1 mg/mL Liberase TM, Sigma‐Aldrich), as previously described (Crocini et al33). Cells were gradually readapted to calcium by adding 50 or 100 μmol/L CaCl₂ every 5 to 8 minutes, until a concentration of 500 μmol/L CaCl₂ was reached.

Peter A.K., Rossi A.C., Buvoli M., Ozeroff C.D., Crocini C., Perry A.R., Buvoli A.E., Lee L.A, & Leinwand L.A. (2019). Expression of Normally Repressed Myosin Heavy Chain 7b in the Mammalian Heart Induces Dilated Cardiomyopathy. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(15), e013318.

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Primary fibroblast isolation protocol

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Primary fibroblasts were isolated from under-arm skin and lung tissues as previously described (Seluanov et al., 2010b ). Briefly, skin tissues were shaved and cleaned with 70% ethanol. Lung tissues were rinsed with PBS to get rid of excess blood. Tissues were minced and incubated in DMEM/F-12 medium (ThermoFisher, 11320033) with Liberase TM (Sigma, 5401127001) at 37°C on a stirrer for 15–90 min. Tis sues were then washed and plated with DMEM/F-12 medium containing 15% fetal bovine serum (Gibco) and Antibiotic-Antimycotic (Gibco). When cells are ~80% confluent, isolated cells were frozen in liquid nitrogen within 2 passages. All subsequent cultures were performed in EMEM (ATCC, 30–2003) supplemented with 15% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco). All primary cells were cultured at 37°C wi th 5% CO₂ and 3% O₂ except naked mole-rat cells, which were cultured at 32°C with 5% CO ₂ and 3% O₂.

Tian X., Firsanov D., Zhang Z., Cheng Y., Luo L., Tombline G., Tan R., Simon M., Henderson S., Steffan J., Goldfarb A., Tam J., Zheng K., Cornwell A., Johnson A., Yang J.N., Mao Z., Manta B., Dang W., Zhang Z., Vijg J., Wolfe A., Moody K., Kennedy B., Bohmann D., Gladyshev V.N., Seluanov A, & Gorbunova V. (2019). SIRT6 is Responsible for More Efficient DNA Double-Strand Break Repair in Long-Lived Species. Cell, 177(3), 622-638.e22.

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Isolation of Mouse Brain Immune Cells

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Mice were anesthetized with Avertin^TM and underwent intra-cardic perfusion with 10 mL of cold PBS. The brain was separated from the brainstem, and the cerebellum was separated from the remaining brain and processed in parallel. The tissue was minced with scissors, placed in digestion buffer (RPMI, 20mM HEPES, 10% FCS, 1mM CaCl2, 1mM MgCl2, 0.05mg/ml Liberase TM (Sigma), 0.02mg/ml DNase I (Sigma)), and incubated for 40 min at 37 °C with shaking at 200 rpm. The mixture was passed through a 100 μm strainer and washed with FACS Buffer (PBS, 1% FBS). Cells were resuspended in 4 mL of 40% Percoll (GE Healthcare) and overlaid on 4 mL of 67% Percoll. Gradients were centrifuged at 400 xg for 20 min at 4 °C and cells at the interface were collected, washed with 10 mL of FACS Buffer, and stained for flow cytometric analysis.

Tang A.T., Choi J.P., Kotzin J.J., Yang Y., Hong C.C., Hobson N., Girard R., Zeineddine H.A., Lightle R., Moore T., Cao Y., Shenkar R., Chen M., Mericko P., Yang J., Li L., Tanes C., Kobuley D., Võsa U., Whitehead K.J., Li D.Y., Franke L., Hart B., Schwaninger M., Henao-Mejia J., Morrison L., Kim H., Awad I.A., Zheng X, & Kahn M.L. (2017). Endothelial TLR4 and the microbiome drive cerebral cavernous malformations. Nature, 545(7654), 305-310.

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Single-Cell RNA Sequencing of Lung and Lymph Node

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Lung sections and mediastinal lymph nodes were taken at the time of necropsy of the animals and processed. Cell suspensions were generated by manually dicing tissue, enzymatically digesting in RPMI containing 0.1 mg/ml Liberase TM (Sigma 5401127001) and 0.02 mg/ml DNaseI (Sigma 11284932001) at 37°C, and then passing through a 100 um filter. Suspensions were subjected to ACK lysis and final washes in PBS containing 0.1% MACS BSA (Miltenyi 130-091-386). 10,000 cells were prepared for 10X Genomics gel bead emulsions. The 10X genomics version 3.0 chemistry was used. cDNA for the individual cells was generated and libraries prepped according to the manufactures protocol. After the final libraries were generated, samples were inactivated for any potentially remaining virus using 500ul of AVL with 500ul of ethanol with a sample volume of 140ul. After a minimum 10-minute incubation, samples were removed from the high-containment laboratory following standard protocols and the libraries were extracted from the AVL using the Qiagen AllPrep DNA spin columns (Cat 80204). Samples were then sent for quantification and sequencing. Samples were sequenced on the NextSeq550 using the 10X suggested cycling.

Speranza E., Williamson B.N., Feldmann F., Sturdevant G.L., Pérez- Pérez L., Mead-White K., Smith B.J., Lovaglio J., Martens C., Munster V.J., Okumura A., Shaia C., Feldmann H., Best S.M, & de Wit E. (2020). SARS-CoV-2 infection dynamics in lungs of African green monkeys. bioRxiv.

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Isolation of Mouse Brain Immune Cells

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Dissociation of Dorsal Root Ganglion Neurons

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Dorsal root ganglion neurons were dissociated and prepared from adult mice using a similar protocol as previously described (Su et al., 2019 (link)). Briefly, mice were sacrificed by exposure to CO₂ and decapitated. DRG were rapidly removed and placed in Puck’s solution containing digesting enzymes. The DRGs were digested with Liberase TM (0.35 U/ml; Sigma-Aldrich, St. Louis, MO, United States) for 35–40 min before another 10 min with Liberase TL (0.25 U/ml; Sigma-Aldrich, St. Louis, MO, United States) and papain (30 U/ml, Worthington Biochemical) at 37°C. The ganglia were then triturated with fire-polished Pasteur pipettes. The dispersed cells were resuspended in F12 (Thermo Fisher Scientific, Waltham, MA, United States) medium supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, United States) and 1% penicillin/streptomycin (MediaTech, Inc., Manassas, VA, United States) and plated on coverslips coated with polyornithine (Neuvitro Corporation, Vancouver, WA, United States) and laminin. Cell cultures were maintained in regular 95% air and 5% CO₂ at 37°C in an incubator.

Wu B., Su X., Zhang W., Zhang Y.H., Feng X., Ji Y.H, & Tan Z.Y. (2021). Oxaliplatin Depolarizes the IB4– Dorsal Root Ganglion Neurons to Drive the Development of Neuropathic Pain Through TRPM8 in Mice. Frontiers in Molecular Neuroscience, 14, 690858.

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Isolation of Lymph Nodes and Skin Cells

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LNs and ear skin were digested as described previously (Gray et al., 2013 (link)). LNs were digested with rotation for 20–30 min at 37°C with 67 µg/ml Liberase TM (Roche Applied Science) and 20 µg/ml DNaseI (Sigma-Aldrich). Ears were split into dorsal and ventral halves and were digested in 2 mg/ml Dispase (Gibco). Separated epidermal and dermal sheets were digested for 60–120 min at 37°C, with rotation, in DMEM containing penicillin–streptomycin, Hepes buffer, 85 µg/ml Liberase TM, 100 µg/ml DNaseI, 0.5 mg/ml hyaluronidase (Sigma-Aldrich), and 2% FCS. Alternatively, whole ear skin was minced and digested with liberase, DNase I, and hyaluronidase as described above. Digestion enzymes were quenched by the addition of 5 mM EDTA and 1% serum. All tissues were disaggregated by passage through a 70-µm or 100-µm nylon sieve (BD Bioscience).

Laidlaw B.J., Gray E.E., Zhang Y., Ramírez-Valle F, & Cyster J.G. (2019). Sphingosine-1-phosphate receptor 2 restrains egress of γδ T cells from the skin. The Journal of Experimental Medicine, 216(7), 1487-1496.

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