Liberase solution

Manufactured by Roche

Sourced in Switzerland

Liberase solution is a laboratory reagent used for the enzymatic dissociation of tissues. It contains a proprietary blend of proteolytic enzymes that can effectively break down the extracellular matrix, allowing for the isolation of individual cells from tissue samples.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Anti cd16 cd32, by BD (2 mentions) Accuri6 flow cytometer, by BD (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Cryovial, by Thermo Fisher Scientific (1 mentions) Serological pipettes, by Corning (1 mentions) Controlled rate freezer, by Thermo Fisher Scientific (1 mentions) Cryostor cs10, by BioLife Solutions (1 mentions) Liberase, by Roche (1 mentions) Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions) Cd3 clone 145 2c11, by BD (1 mentions) Dnase 1, by New England Biolabs (1 mentions) 70 μm strainer, by BD (1 mentions) Cacl2 mgcl2, by Thermo Fisher Scientific (1 mentions) Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions) 30 μm pre separation filter, by Miltenyi Biotec (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Iscove s modified dulbecco s medium, by Corning (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Tgf β, by Thermo Fisher Scientific (1 mentions)

11 protocols using liberase solution

Liver Nonparenchymal Cell Isolation

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Livers were perfused through the portal vein with a 30 μg/ml liberase solution (Roche), mechanically disrupted, and digested for 10 min at 37 °C in a 30 μg/ml liberase solution with 10 μg/ml of DNAse I (Roche) and 200 μg/ml pronase (Roche), then filtered through a 100-μm cell strainer. Parenchymal cells were separated from NPC by centrifugation for 5 min at 60×g. The supernatant was collected and centrifuged for 10 min at 420×g. The pellet containing the NPC was resuspended in a 40% percoll solution (GE Healthcare), laid onto an 80% percoll solution. After centrifugation for 20 min at 800×g at room temperature, the NPC fraction was collected at the interface and subjected to flow cytometric analysis or cell sorting.

Deust A., Chobert M.N., Demontant V., Gricourt G., Denaës T., Thiolat A., Ruiz I., Rodriguez C., Pawlotsky J.M, & Teixeira-Clerc F. (2021). Macrophage autophagy protects against hepatocellular carcinogenesis in mice. Scientific Reports, 11, 18809.

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Cryopreservation of iPSC-derived Cardiomyocytes

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iPSC‐derived cardiomyocytes were collected from the bioreactor on Day 14, washed with phosphate buffered saline (PBS)^−/− (ThermoFisherScientific) once and resuspended in Liberase solution (50 μg/ml; Roche) and incubated at 37°C for 30 min. The aggregates were triturated every 15 min using 10 ml serological pipettes (Corning). Following dissociation, an equal volume of growth medium (RPMI‐1640 + B27 with insulin) was added to the cell suspension to dilute the Liberase. The cells were centrifuged at 200g for 3 min at room temperature. Following centrifugation, the supernatant was removed and discarded. TrypLE solution was added to the cells and incubated for 7–8 min at 37°C. The enzyme was subsequently diluted by adding equal volume of growth medium. Viable cell counts were performed with the NC‐200 and the cells centrifuged at 200g for 3 min at 4°C. The supernatant was removed, discarded and the cell pellet was resuspended in CryoStor CS10 (BioLife Solutions) supplemented with 10 μM Y‐27632 at 5 × 10⁶ cells/ml. Cryovials (ThermoFisherScientific) were filled at 1.0 ml and cryopreserved using a Controlled Rate Freezer (ThermoFisherScientific).

Shafa M., Panchalingam K.M., Walsh T., Richardson T, & Baghbaderani B.A. (2019). Computational fluid dynamics modeling, a novel, and effective approach for developing scalable cell therapy manufacturing processes. Biotechnology and Bioengineering, 116(12), 3228-3241.

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Isolating and Culturing Drosophila Neurons

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The protocol is modified based on the protocol from the Sprecher lab (Egger et al., 2013 (link)). Drosophila third-instar larvae were picked and washed with distilled H₂O, 70% EtOH, and sterile 1× modified dissection saline (9.9 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 137 mM NaCl, 5.4 mM KCl, 0.17 mM NaH₂PO₄, 0.22 mM KH₂PO₄, 3.3 mM glucose, 43.8 mM sucrose). Optic lobes and ventral ganglion were dissected and cleaned in sterile 1× modified dissection saline; tissues were then dissociated in Liberase solution (Roche, Basel, Switzerland; Liberase TM Research Grade, 05401119001, stock solution 2.5 mg/ml 13 Wünsch units/ml in sterile 1× modified dissection saline, final concentration in 0.25 mg/ml 1.3 Wünsch units/ml) for 1 h. The neurons were spun down at 300 × g for 5 min and then washed twice in supplemented Schneider's medium (20% fetal bovine serum, 5 μg/ml insulin, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml tetracycline); the neurons were plated onto concanavalin A–coated coverslips and allowed attached for 30 min before addition of full volume of supplemented Schneider's medium for imaging 2–4 h after plating or incubated for 2–3 d before imaging.

Lu W., Lakonishok M, & Gelfand V.I. (2015). Kinesin-1–powered microtubule sliding initiates axonal regeneration in Drosophila cultured neurons. Molecular Biology of the Cell, 26(7), 1296-1307.

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Cardiac Cell Isolation from Mouse Hearts

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In brief, mice were anaesthetized with 3% isoflurane and killed by cervical dislocation. Hearts were quickly excised and fresh cardiac tissues were divided into 1 mm³ pieces and digested by 0.5 mg/mL liberase solution (Roche, 05401020001) at 37 °C for 20 minutes to generate single-cell suspensions. Afterwards, heart tissue pieces were gently shaken in a 37 °C shaker to guarantee complete digestion of tissues. After the digestive process was completed, the supernatant medium was harvested and the same volume of Dulbecco's Modified Eagle's medium (DMEM, ATCC, 302002) containing 10% fetal bovine serum (FBS, Gibco, 10099141) was added to abort the reaction. A New liberase solution was then added to the surplus tissue fragments for further digestion. To obtain single cell suspension, the mixture of supernatant and neutralization solution was subsequently passed through a 40-μm cell strainer. The collected cells were centrifuged for 5 minutes at the speed of 300 g/min at 4°C. Subsequently, cell pellets were suspended in relevant neutralization buffer for further experiments.

Gong H., Wang T., Sun X., Zhang Y., Qiu Y., Sun W., Zhang Y., Teng P., Hu Y., Hu X., Ma L., Xu Q, & Zhao H. (2024). Fibroblasts facilitate lymphatic vessel formation in transplanted heart. Theranostics, 14(5), 1886-1908.

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Evaluating Immune Cell Populations in Spleen and Heart

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Spleen and heart fragments were collected to evaluate IFN-γ-producing CD4⁺ T cells, IL-10-producing Tregs, and IL-10-producing Tr1 cells. To isolate mononuclear cells from cardiac tissues, the hearts were removed at 15 d.p.i. washed (to remove blood clots), minced with scissors into small fragments, extensively washed, and subjected to enzymatic digestion with 500 mg/mL of Liberase solution (Roche Applied Science, Indianapolis, IN, USA) for 1 h at 37°C. The tissues were washed with RPMI 10% FCS and total cells that passed through the cell strainer were subsequently counted. Two million leukocytes from heart tissue or from spleen were stimulated in vitro with PMA/Ionomycin/Golgi Stop for 6 h. The leukocytes were stained with specific antibodies for Cd11b (clone MI/70 BD), I-A/E (clone 269 BD), CD3 (clone 145-2C11), CD4 (clone RM4-5 BD), Foxp3 (clone MF 23 BD), IFN-γ (clone XMG 1.2 BD), IL-10 (clone JESS-16E3 BD), and IL-27p28 (clone MM27-7b1 Biolegend), and evaluated by flow cytometry.

Medina T.S., Oliveira G.G., Silva M.C., David B.A., Silva G.K., Fonseca D.M., Sesti-Costa R., Frade A.F., Baron M.A., Ianni B., Pereira A.C., Chevillard C., Cunha-Neto E., Marin-Neto J.A, & Silva J.S. (2017). Ebi3 Prevents Trypanosoma cruzi-Induced Myocarditis by Dampening IFN-γ-Driven Inflammation. Frontiers in Immunology, 8, 1213.

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Tumor Dissociation and Single-Cell Isolation

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Primary and secondary tumors were weighed and digested for 20 min at 37 °C in 5 ml PBS plus MgCl₂/CaCl₂ (Gibco; Thermo Fisher Scientific) supplemented with 50 μg/mL DNase I (New England BioLabs) and 120 μg/mL Liberase solution (Roche Life Science). After the incubation, tumor pieces were mechanically ground through a 70-μm strainer (Falcon) and filtered through a 30-μm pre-separation filter (Miltenyi Biotech). Lymph nodes were squeezed through a 70-μm strainer. Red blood cell (RBC) lysis was performed using 1× RBC lysis buffer (eBioscience).

Onyshchenko K., Luo R., Rao X., Zhang X., Gaedicke S., Grosu A.L., Firat E, & Niedermann G. (2024). Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models. Theranostics, 14(6), 2573-2588.

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Isolation and TGF-β Treatment of Inflammatory Myeloid Cells and Cardiac Fibroblasts

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Inflammatory myeloid cells (referred to earlier as prominin-1⁺/CD133⁺ progenitors) were obtained as described previously [8 (link),29 (link)] with minor modifications. Briefly, myocarditis-positive hearts at day 17–21 of EAM were perfused and dissected into pieces and incubated in the Liberase solution (Roche, Basel, Switzerland) for 45–60 min at 37 ˚C. Cellular suspensions were filtered through 70 µm and 40 µm cell strainers and plated in the culture medium containing Iscove’s modified Dulbecco’s medium (Corning, New York, NY, USA) supplemented with 20% foetal bovine serum (FBS, Gibco, Waltham, WA, USA), penicillin–streptomycin (1:100, Gibco) and β-mercaptoethanol (1:1000, Sigma-Aldrich, Saint Louis, USA). Cardiac fibroblasts were isolated from hearts of healthy mice and cultured in the Dulbecco’s Modification of Eagle’s Medium supplemented with 10% FBS (Gibco), penicillin–streptomycin (1:100, Gibco) and β-mercaptoethanol (1:1000, Sigma). For experiments, cells at the first or second passage were used. Both types of cells were treated with 10 ng/mL TGF-β (PeproTech, London, UK) for 30 min, 1 h, 6 h, 24 h and 3 days before harvesting. Control cells were cultured without TGF-β.

Tkacz K., Rolski F., Czepiel M., Działo E., Siedlar M., Eriksson U., Kania G, & Błyszczuk P. (2020). Haploinsufficient Rock1+/− and Rock2+/− Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis. Cells, 9(3), 700.

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Isolation and Flow Cytometry of Mouse Lung Cells

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Single cell suspensions from bronchoalveolar lavage fluid or from mouse lung isolates were subjected to flow cytometry. To isolate single-cell suspensions from mouse lung, the lungs were minced and incubated in 50 μg/ml Liberase solution (Roche) for 30 min at 37C and agitated at 750rpm. The cells were then disassociated with an 18-gauge needle. The tissue digest was passed through a 40μm cell strainer into a tube with wash buffer and centrifuged at 400 x g, 4°C for 5 min. The pellet was then re-suspended in 50 mL 1% BSA (VWR Life Sciences) in PBS and centrifuged at 400 x g, 4°C for 5 min and the supernatant was discarded. The cell pellet was re-suspended at 2.5 × 10⁷ cells/mL in FACS buffer. Single cell suspensions were then incubated with anti-CD16/CD32 (BD Bioscience) to block Fc receptor binding (20 min, 4 °C). Cells were pelleted by centrifugation and re-suspended in primary fluorochrome-conjugated antibodies (see above in ice-cold FACS buffer. After washing with 1% BSA in PBS, at least 100,000 live cells per sample were collected for analysis on a BD Accuri6 flow cytometer, in which neutrophils were counted as Ly6G and CD11b double positive after excluding dead cells which were 7-AAD positive. Data analysis was performed using FlowJo software (FlowJo, LLC) as in (31 (link)).

Jia H., Sodhi C.P., Yamaguchi Y., Lu P., Martin L.Y., Good M., Zhou Q., Sung J., Fulton W.B., Nino D.F., Prindle T J.r., Ozolek J.A, & Hackam D.J. (2016). Pulmonary Epithelial Toll-like Receptor 4 Activation leads to Lung Injury in Neonatal Necrotizing Enterocolitis. Journal of immunology (Baltimore, Md. : 1950), 197(3), 859-871.

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Isolation and Culture of Murine Cardiac Fibroblasts

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Murine cardiac fibroblasts were also isolated from untreated C57BL/6J mice, as previously described [34 (link)]. Briefly, five mice per isolation were euthanised (sodium pentobarbitone, 200 mg/kg i.p.) and heparinised before their hearts were removed and ventricles isolated prior to mincing and mixing with 4 mL Liberase™ solution (Roche, Burgess Hill, UK) at 37 °C for 8 min. The supernatant was then removed and the process was repeated a further three times with the undigested tissue prior to filtration, centrifugation, and resuspension of the pellet in supplemented DMEM (10 % FBS, 20 mmol/L l-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin). Cells were then transferred to a 1 % gelatin-coated T75 flask with 10 mL DMEM at 37 °C and cultured in a humidified atmosphere of 5 % CO₂ for approximately 7 days until 90 % confluent, when they were trypsinised, centrifuged, and reseeded at 1:2 for expansion. At passage 2, cardiac fibroblasts were treated with conditioned media harvested from BMDM (as detailed above), with or without TGF-β (5 ng/mL, to induce myofibroblast differentiation), prior to preparation of RNA/cDNA and real-time RT-PCR analysis of mRNA expression, as described previously.

Tate M., Robinson E., Green B.D., McDermott B.J, & Grieve D.J. (2015). Exendin-4 attenuates adverse cardiac remodelling in streptozocin-induced diabetes via specific actions on infiltrating macrophages. Basic Research in Cardiology, 111, 1.

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Flow cytometry of mouse lung cells

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Flow cytometry was performed in single cell suspensions from mouse lung as reported^{(12 (link))} before and in brief as follows: freshly harvested sections of mouse lung were minced and incubated in 50 μg/ml Liberase solution (Roche) for 30 min at 37°C and agitated at 750rpm. The cells were then disassociated with an 18-gauge needle. The tissue digest was passed through a 40 μm cell strainer into a tube with wash buffer and centrifuged at 400 × g, 4°C for 5 min. The pellet was then re-suspended in 50 mL 1% BSA (VWR Life Sciences) in PBS and centrifuged at 400 × g, 4°C for 5 min and the supernatant was discarded. The cell pellet was re-suspended at 2.5 × 10⁷ cells/mL in FACS buffer. Single cell suspensions were then incubated with anti-CD16/CD32 (BD Bioscience) to block Fc receptor binding (20 min, 4 °C). IL-17A⁺ and Foxp3⁺ cells were analyzed according to manufacturer’s guidance on a BD Accuri6 flow cytometer.

Jia H., Sodhi C.P., Yamaguchi Y., Lu P., Ladd M.R., Werts A., Fulton W.B., Wang S., Prindle T J.r, & Hackam D.J. (2019). Toll Like Receptor 4 Mediated Lymphocyte Imbalance Induces NEC-Induced Lung Injury. Shock (Augusta, Ga.), 52(2), 215-223.

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