Liberase dl

Manufactured by Merck Group

Sourced in United States

Liberase DL is a laboratory reagent produced by Merck Group for the gentle dissociation and isolation of cells from tissue samples. It is a highly purified enzyme solution that contains a proprietary blend of collagenases and other enzymes designed to facilitate the effective and gentle disruption of extracellular matrix components.

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Liberase (268 citations)

25 protocols using liberase dl

Isolation and Purification of Murine Bone Marrow Stromal Cells

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Mice were euthanized according to standardized and approved IACUC protocols. Femurs and tibiae were isolated, cleaned of soft tissue, cut at both ends, and marrow centrifuged out of the diaphyses and metaphyses into a collection tube. Marrow was resuspended in 1 mg/mL Liberase DL (Sigma) diluted in FACS buffer (0.5% BSA [Sigma] in PBS) and digested at 37 °C for 30 min to increase yield of stromal cells released from the vasculature fraction as previously described^{33 (link)}. Diaphyses and metaphyses cleared of bone marrow were gently crushed, rinsed in PBS, and then digested in 300 Units/mL of Collagenase IA (Sigma), diluted in MEM α (ThermoFisher), 3 times for 25 min each. Bone and marrow solutions were then combined and treated with RBC lysis buffer (ThermoFisher) to clear erythrocytes. The sample was then depleted of cells expressing hematopoietic lineage markers (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7–4, and Ter-119) using Magnet Assisted Cell Sorting (MACS) and the Lineage Cell Depletion Kit (Miltenyl Biotec).

Doolittle M.L., Saul D., Kaur J., Rowsey J.L., Vos S.J., Pavelko K.D., Farr J.N., Monroe D.G, & Khosla S. (2023). Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton. Nature Communications, 14, 4587.

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Isolation and Culture of Murine Fibroblasts

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Wild-type, Bth/+ and Bth/Bthfibroblasts were obtained from P5 pups. Mice were euthanized and cleaned with
70% ethanol. 1-2 cm² underarm skin fragments were excised and
immerged in cold HBSS (ThermoFisher). Subcutaneous fat was removed by forceps.
Skin fragments were cut to ~1 mm² pieces with a 25G
5/8″ Syringe (1180125058, Covidien). Tissues were digested with 0.5
mg/mL Liberase DL (Sigma 5401160001) at 37 °C for 1 hour with occasional
pipetting up and down to break cell clumps. Warm culture medium (1:1 DMEM:F12
media (ThermoFisher) with 15% FBS (ThermoFisher) and 100 U/mL
penicillin+streptomycin (ThermoFisher) was added to stop the enzyme
digestion. The solution was filtered with a 70-μm cell strainer (Falcon)
and centrifuged at 200 g for 5 min. The pellet was resuspended in culture medium
and transferred to a 25-mL culture flask, then incubated at 37°C,
5% CO₂, 3% O₂. Fibroblasts were cultured
for 2 ~ 3 days to reach ~90% confluence, then passaged
in 100-mL flasks in DMEM plus GlutaMax (ThermoFisher) supplemented with
10% (v/v) fetal bovine serum (FBS) at 37 °C with 5%
CO₂.

Gao X., Tao Y., Lamas V., Huang M., Yeh W.H., Pan B., Hu Y.J., Hu J.H., Thompson D.B., Shu Y., Li Y., Wang H., Yang S., Xu Q., Polley D.B., Liberman M.C., Kong W.J., Holt J.R., Chen Z.Y, & Liu D.R. (2017). Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature, 553(7687), 217-221.

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Isolation and Purification of Bone Marrow Endothelial Cells

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Bone marrow endothelial cells (ECs) were stained by intravenous injection of 10 μg anti-VE-cadherin antibody (BD Biosciences, PE Rat Anti-Mouse CD144, #562243) 5-10 min before euthanizing the mice. Tibias and femurs were gently crushed using a mortar and pestle and then digested with DNase I (200 U/mL; ThermoFisher, # EN0521), Liberase^DL (250 mg/mL; Sigma, # 5401160001), type IV collagenase (1 mg/mL; ThermoFisher, # 17104019), and collagenase D (500 μg/mL; Sigma, # 11088858001) with agitation for 30 min at 37 °C. The cells were dissociated to a single-cell suspension by passing through a 25G needle several times and filtered with a 70-µm nylon mesh to generate a single-cell suspension. Cells were sorted in two successive rounds to ensure high purity using a FACS Aria II flow cytometer.

Zhao Q., Han Y.M., Song P., Liu Z., Yuan Z, & Zou M.H. (2022). Endothelial cell-specific expression of serine/threonine kinase 11 modulates dendritic cell differentiation. Nature Communications, 13, 648.

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Isolation and Purification of Lymphocytes

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Mice were sacrificed with isoflurane, and cardiac puncture was performed prior to organ removal. Spleens were processed with a GentleMACS Dissociator (Miltenyi Biotec), filtered through nylon mesh, and layered onto a Lympholyte-M gradient (Cedarlane Laboratories Ltd., Canada). Lymphocytes were harvested from the gradient interface, and washed once in PBS supplemented with 1% FBS (1% PBS-serum). Alternatively, spleens were processed with ammonium chloride to lyse red blood cells and enrich for lymphocytes. Livers were perfused with 1% PBS-serum before removal, processed in 1% PBS-serum with the GentleMACS, and filtered through nylon mesh. Samples were washed 3 times with 1% PBS-serum, suspended in 40% Percoll and layered on 70% Percoll. Lymphocytes were harvested from the gradient interface and washed once with 1% PBS-serum. Extraorbital lacrimal glands were processed in Collagenase IV (Sigma-Aldrich) or Liberase-DL (Sigma-Aldrich) with the GentleMACS, incubated at 37 °C for 10 minutes, filtered through nylon mesh, and washed once with 1% PBS-serum before being layered on a Lympholyte-M gradient. In some experiments, 6 to12 lacrimal glands from 3 to 6 animals were pooled. Lymphocytes were harvested from the gradient interface and washed once in 1% PBS-serum.

Erick T., Grigoryan L, & Brossay L. (2017). Lacrimal Gland NK Cells Are Developmentally and Functionally Similar to Conventional NK Cells. ImmunoHorizons, 1(2), 2-9.

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Brain Vessel Purification from Mice

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Mechanical purification of brain vessels from 2-month-old mice was performed as previously described [19 ]. A volume of 100 μl ml⁻¹ cycloheximide was added to all buffers when using Aldhl1:L10a-eGFP mice (see below). Brain vessels submitted to basal lamina digestion were obtained replacing brain mechanical homogenization by a 45 min digestion with DNAseI (Sigma, St Louis, MO, USA) (20 U ml⁻¹) and Liberase DL (Dispase Low, Sigma; 37.5 μg ml⁻¹) in HBSS (Life Technology) at 37 °C.

Boulay A.C., Saubaméa B., Adam N., Chasseigneaux S., Mazaré N., Gilbert A., Bahin M., Bastianelli L., Blugeon C., Perrin S., Pouch J., Ducos B., Le Crom S., Genovesio A., Chrétien F., Declèves X., Laplanche J.L, & Cohen-Salmon M. (2017). Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface. Cell Discovery, 3, 17005-.

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Tissue Preparation for FACS Analysis

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Preparation of animal tissue for FACS was performed as previously described.¹⁶ In brief, wounds were harvested (n = 6 wounds per timepoint) and minced with fine scissors before adding to 0.5mg/mL Liberase DL (Sigma-Aldrich) in Dulbecco’s modified Eagle Medium (DMEM, Gibco), then incubated at 37°C for 30 minutes. After digestion, enzyme was quenched by addition of media containing 20% fetal bovine serum (FBS, Gibco), and tissue was strained through a 100 μm followed by a 40 μm strainer to achieve a single cell suspension. Cells were incubated with preconjugated antibodies for 30 minutes in PBS with 1% FBS, 0.1% penicillin/ streptomycin (P/S, Gibco), and 1 mM ethylenediaminetetraacetic acid (Invitrogen). Antibodies are listed in Supplemental Table 2, http://links.lww.com/SLA/B552. Cells were washed with PBS to remove antibodies in solution before performing flow cytometry.

Moore A.L., desJardins-Park H.E., Duoto B.A., Mascharak S., Murphy M.P., Irizarry D.M., Foster D.S., Jones R.E., Barnes L.A., Marshall C.D., Ransom R.C., Wernig G, & Longaker M.T. (2020). Doxycycline Reduces Scar Thickness and Improves Collagen Architecture. Annals of surgery, 272(1), 183-193.

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Isolation of Exosomes from Murine Tuberculosis

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BALB/c mice (6 to 8 weeks old) were infected with M. tuberculosis 100 bacilli per lung by aerosol or left uninfected as described previously (100 (link)). At 20 weeks p.i., animals were euthanized and serum and lungs were collected. Exosomes were isolated from mouse serum by precipitation in ExoQuick Ultra solution overnight per the manufacturer’s instructions.
To isolate exosomes from lungs, 2 ml of tissue digestion mix (serum-free RPMI 1640 with 200 μg/ml Liberase DL [Sigma-Aldrich] and 100 μg/ml of DNase [Thermo Fisher Scientific]) was added to one whole lung and transferred to C-tubes. Lungs were homogenized on a gentleMACS Dissociator (Miltenyi Biotec). Samples were incubated for 30 min at 37°C at 70 to 100 rpm followed by further homogenization. Lung homogenates were passed through a 40-μm-pore-size cell strainer and centrifuged at 1,500 rpm for 5 min. Supernatants were collected and passed through a 0.22-μm-pore-size filter. Filtered supernatants were concentrated to 1 ml, and exosomes were isolated using ExoQuick Ultra precipitation solution as described above. Exosomes were stored at −80°C for future analysis.

Tyagi P., Pal V.K., Agrawal R., Singh S., Srinivasan S, & Singh A. (2020). Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics. mBio, 11(2), e03293-19.

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Isolation and Culture of Murine Fibroblasts

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Metastatic Tumor Dissection and Isolation

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Lungs were perfused with PBS through the right ventricle, and metastases (<1 mm in diameter) were dissected under a dissection microscope and pooled from 1–5 mice. The tissues were subsequently digested using an enzyme mix of Liberase DL (Sigma-Aldrich 5466202001, 0.52 U/mL), TL (Sigma-Aldrich 5401020001, 0.26 U/mL) and DNase I (Sigma-Aldrich DN25, 150 µg/mL) for 30 min at 37°C. For sorting for RNA-seq or qRT-PCR, transcription inhibitors alpha-amanitin (Sigma-Aldrich A2263, 5 µg/mL) and actinomycin D (Sigma-Aldrich A1410, 1 µg/mL) were also added to the digestion buffer. Flow cytometry was analyzed using a LSRII cytometer (BD Biosciences), and the data were analyzed using Flowjo software (TreeStar, v10; a possible free alternative is Flowing Software 2.5.1 from University of Turku). FACSAria II (BD Biosciences) and Moflo Astrios (Beckman Coulter) were used for sorting. Antibody information is found in
Table 1.

Zheng W., Zhao D., Zhang H., Chinnasamy P., Sibinga N, & Pollard J.W. (2021). Induction of interferon signaling and allograft inflammatory factor 1 in macrophages in a mouse model of breast cancer metastases. Wellcome Open Research, 6, 52.

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Tracking Lymph Node Immune Responses

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Distribution was measured by cellular GFP expression with EGFP-encoding mRNA. BALB/c mice (n = 4/group, 5 weeks old) were immunized intramuscularly with saline or LNP-encapsulated EGFP mRNA containing L202 at a dose of 10 μg of mRNA. After 24 h, mouse inguinal lymph nodes were collected and digested with RPMI-1640 medium (Wako #189-02025) containing 0.3 mg/mL Liberase DL (Sigma-Aldrich #05401160001) and 0.2 mg/mL DNase I (Roche #10104159001) at 37°C for 30 min and then filtered through a 70-μm-mesh cell strainer (Corning #352350). Cells were pre-incubated with Mouse BD Fc Block (BD Biosciences #553142) for 10 min and stained with fluorophore-conjugated antibodies as follows: anti-CD45 (clone: 3F-11, BD Biosciences #565967), B220 (clone: RA3-6B2, BioLegend #103210), CD3ε (clone: 17A2, BD Biosciences #612803), CD11b (clone: M1/70, BD Biosciences #624294), CD11c (clone: N418, BioLegend #117339), IA/IE (clone: M5/114.15.2, BD Biosciences #5660866), CD317 (clone: 129C1, BD Biosciences #127025), Ly6C (clone: HK1.4, BD Biosciences #128044), Ly6G (clone: 1A8, BD Biosciences #560601), and CD169 (clone: 3D6.112, BioLegend #142421). Data acquisition was performed on a BD FACSymphony Flow Cytometer (BD Biosciences) and data were analyzed by FlowJo software (BD Biosciences).

Suzuki Y., Miyazaki T., Muto H., Kubara K., Mukai Y., Watari R., Sato S., Kondo K., Tsukumo S.I., Yasutomo K., Ito M, & Tsukahara K. (2022). Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates. Molecular Therapy. Nucleic Acids, 30, 226-240.

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