DRGN were isolated from rat neonates (P1–3) in accordance with the Charité Universitätsmedizin Berlin and state authority animal care committee’s regulations. Cells were digested in 0.28 Wünsch unit collagenase (Liberase DL, Roche, Germany) and separated by gentle trituration. Afterward, cells were passed through a 70 µm cell strainer and centrifuged with a Percoll gradient (1.019/1.038 g/ml) at 1000 g for 10 min. This typically yields a higly enriched DRGN fraction with <10% contaminating cells17 (link). After centrifugation, cells were plated on poly-L-lysine/laminin-coated coverslips and maintained in Neurobasal-A media supplemented with B-27 (Gibco, Darmstadt, Germany), 0.5 mM glutamine/fresh nerve growth factor (10 ng/ml) and incubated overnight in a 95% air/5% CO2 humidified atmosphere in an incubator at 37 °C before treatment.
Liberase dl
Manufactured by Roche
Sourced in Germany, Switzerland
Liberase DL is a digestive enzyme blend developed by Roche for the dissociation of tissue samples. The product is designed to effectively break down the extracellular matrix and facilitate the isolation of cells from a variety of tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (9 mentions)
Dnase 1, by Roche (8 mentions)
Dnase, by Merck Group (4 mentions)
Hanks balanced salt solution (hbss), by Merck Group (4 mentions)
Liberase tl, by Roche (3 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (3 mentions)
Dispase 2, by Roche (2 mentions)
Bovine serum albumin (bsa), by Roche (2 mentions)
Leupeptin, by Roche (2 mentions)
Dnase, by Roche (2 mentions)
Amplicor hiv 1 monitor ultrasensitive method, by Roche (2 mentions)
Gentlemacs, by Miltenyi Biotec (2 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Neurobasal a media, by Thermo Fisher Scientific (1 mentions)
Soybean trypsin inhibitor, by Roche (1 mentions)
Easysep mouse monocyte enrichment kit, by STEMCELL (1 mentions)
Rbc lysis buffer solution, by Thermo Fisher Scientific (1 mentions)
Mcp 1, by R&D Systems (1 mentions)
Easysep t cell enrichment kit, by STEMCELL (1 mentions)
68 protocols using liberase dl
1
Isolation of Neonatal Rat Dorsal Root Ganglion Neurons
2
Isolation and Enrichment of Tumor Stem Cells
3
Isolation and Culture of PBMC and RMMC
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Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque ™ (Pfizer-Pharmacia, New York, NY) and rested overnight in R-15 at 37°C, 5% CO2. Rectal mucosal mononuclear cells (RMMCs) were isolated from biopsies using enzymatic and mechanical disruption as previously described48 (link). Briefly, biopsies were subjected to shaking incubation at 37°C for 30 minutes in 25mg/mL Liberase DL (Roche, Indianapolis, IN) followed by passage through a 16-gauge blunt end needle to disrupt tissue. Following disruption, free cells were collected through a sterile 70µm cell strainer. The disruption process was repeated until all biopsy tissue was digested. Free RMMCs were washed three times in 20mL of R-15 and rested overnight at 37°C, 5% CO2 in R-15 supplemented with 200× Zozyn (Pfizer-Pharmacia).
4
Isolation and Characterization of Cardiomyocytes
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The wash solution consisted of (in mM) 117 NaCl, 5.7 KCl, 1.5 KH2PO4, 4.4 NaHCO3, 1.7 MgCl2, 21 HEPES, 20 taurine, 11.7 glucose and 10 2, 3-butanedione monoxime. pH was adjusted to 7.4 with NaOH.
For the digestion solution, 0.25 mg/ml Liberase DL (Roche) and 1.36 mg/ml Dispase II (Roche) were added to 20 ml of the wash solution.
For the sedimentation solution, 2 mM pyruvate, 10 μM leupeptin (Roche), 2 μM soybean trypsin inhibitor, and 3 mg/ml BSA (Roche) were added to 40 ml of the wash solution.
For experiments, the following bath solution was used (in mM): 150 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1.13 CaCl2, 10 glucose and 10 HEPES. pH was adjusted to 7.4 with NaOH. The same solution was also used to fill the glass pipettes.
All chemicals were obtained from Sigma-Aldrich if not otherwise mentioned.
For the digestion solution, 0.25 mg/ml Liberase DL (Roche) and 1.36 mg/ml Dispase II (Roche) were added to 20 ml of the wash solution.
For the sedimentation solution, 2 mM pyruvate, 10 μM leupeptin (Roche), 2 μM soybean trypsin inhibitor, and 3 mg/ml BSA (Roche) were added to 40 ml of the wash solution.
For experiments, the following bath solution was used (in mM): 150 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1.13 CaCl2, 10 glucose and 10 HEPES. pH was adjusted to 7.4 with NaOH. The same solution was also used to fill the glass pipettes.
All chemicals were obtained from Sigma-Aldrich if not otherwise mentioned.
5
Monocyte Migration Assay Protocol
6
Isolation of PBMCs and Rectosigmoid Mononuclear Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from blood using Ficoll-Paque™ (Pfizer-Pharmacia, New York, NY) and rested overnight in R-15 at 37°C, 5% CO2. Rectosigmoid mononuclear cells were isolated from biopsies as described46 (link), 47 (link). Briefly, biopsies were washed in R-15 and subjected to shaking incubation in 25mg/mL Liberase® DL (Roche, Indianapolis, IN) at 37°C for 30 minutes, then passed through a 16-gauge blunt end needle to mechanically disrupt tissue. Disrupted tissue was passed through a sterile 70μm cell strainer to collect cells. Undigested tissue was again incubated in Liberase and the process repeated until all tissue was digested. Isolated cells were washed three times in R-15 and rested overnight at 37°C, 5% CO2 in R-15 with 200x Zozyn (Pfizer-Pharmacia).
7
Isolation and Co-culture of Atherosclerotic Plaque Cells
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Atherosclerotic plaques were obtained from patients who were undergoing carotid endarterectomy at the Department of Surgery, Vascular Surgery, Södersjukhuset, Stockholm, Sweden. This study was pre-approved by the research ethics committee of Karolinska Institutet and in accordance with the Declaration of Helsinki. All subjects gave their written informed consent before entering the study.
Cells were isolated from atherosclerotic plaques as described earlier (22) . In brief, the plaques were first dissected into small pieces, which were then incubated with 1.25 mg/ml collagenase IV (Life Technologies Europe BV, Stockholm, Sweden), 25 μg/ml Liberase DL (Roche Applied Science, Stockholm, Sweden), and 0.2 mg/ml DNase I (Roche Applied Science, Stockholm, Sweden) for 1 h at 37°C. The dissociated plaque cells were then passed through a 100-μm Celltrics filter (Millipore AB, Stockholm, Sweden) to remove unwanted fat and debris, and T cells were purified with the EasySep T-cell enrichment kit (STEMCELL Technologies). DCs obtained from the peripheral blood of the plaque donors were treated with or without MDA-HSA as described above and thereafter co-cultured with plaque T cells for 48 h. In addition, plaque T cells were cultured in the presence or absence of MDA-HSA for 24 h.
Cells were isolated from atherosclerotic plaques as described earlier (22) . In brief, the plaques were first dissected into small pieces, which were then incubated with 1.25 mg/ml collagenase IV (Life Technologies Europe BV, Stockholm, Sweden), 25 μg/ml Liberase DL (Roche Applied Science, Stockholm, Sweden), and 0.2 mg/ml DNase I (Roche Applied Science, Stockholm, Sweden) for 1 h at 37°C. The dissociated plaque cells were then passed through a 100-μm Celltrics filter (Millipore AB, Stockholm, Sweden) to remove unwanted fat and debris, and T cells were purified with the EasySep T-cell enrichment kit (STEMCELL Technologies). DCs obtained from the peripheral blood of the plaque donors were treated with or without MDA-HSA as described above and thereafter co-cultured with plaque T cells for 48 h. In addition, plaque T cells were cultured in the presence or absence of MDA-HSA for 24 h.
8
Mouse Lung and Immune Cell Isolation
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Mouse lung tissue was dissociated as previously reported (Patnode et al., 2014 (link)). Lungs were minced and incubated in digestion buffer (0.2 U/ml Liberase DL (Roche Applied Sciences) and 0.2 mg/ml DNase (Sigma) in Hank’s Buffered Salt Solution (without Ca2+/Mg2+) for 25 min at 37°C before being passed through a 70μm cell strainer. Spleen and lymph nodes were dissociated manually and passed through a 70μm cell strainer. Red blood cells were removed from lung and spleen samples by treating with ACK lysis buffer.
9
Cardiac Cell Isolation and Microscopy Solutions
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The wash solution consisted of (in mM) 117 NaCl, 5.7 KCl, 1.5 KH2PO4, 4.4 NaHCO3, 1.7 MgCl2, 21 HEPES, 20 taurine, 11.7 glucose and 10 2,3-butanedione monoxime. pH was adjusted to 7.4 with NaOH.
For the digestion solution, 0.25 mg/ml Liberase DL (Roche) and 1.36 mg/ml Dispase II (Roche) were added to 20 ml of the wash solution.
For the sedimentation solution, two mM pyruvate, 10 μM leupeptin (Roche), two μM soybean trypsin inhibitor, and three mg/ml BSA (Roche) were added to 40 ml of the wash solution.
For the microscopy experiments following solution was used (in mM): 150 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1.13 CaCl2, 10 glucose and 10 HEPES. pH was adjusted to 7.4 with NaOH.
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) if not mentioned otherwise.
For the digestion solution, 0.25 mg/ml Liberase DL (Roche) and 1.36 mg/ml Dispase II (Roche) were added to 20 ml of the wash solution.
For the sedimentation solution, two mM pyruvate, 10 μM leupeptin (Roche), two μM soybean trypsin inhibitor, and three mg/ml BSA (Roche) were added to 40 ml of the wash solution.
For the microscopy experiments following solution was used (in mM): 150 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1.13 CaCl2, 10 glucose and 10 HEPES. pH was adjusted to 7.4 with NaOH.
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) if not mentioned otherwise.
10
Dissection and Isolation of Murine Organ Cells
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We removed each tissue and washed each in HBSS, then dissected the tissue into small pieces using a razor blade. Briefly, kidney and lung were digested using Liberase DL and TM (Roche), respectively. Spleen was mechanically dissociated. Cells were filtered using 40-µm cell strainers and diluted to 106 cells/mL before single-cell library preparation (for details, see Supplemental Methods ).
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