After review and approval by the ethical committee at the University of Heidelberg, we obtained written informed consent from 18 healthy pregnant women, procured umbilical cords immediately after delivery and stored them in PBS at 4°C. We cannulated the umbilical vein on both sides, flushed it with PBS, and filled it with 20 mL of 0.1% collagenase–dispase (Boehringer Mannheim, Mannheim, Germany) in Hanks' balanced salt solution (Gibco, Grand Island, NY, USA). After 20 min of incubation at 37°C, we gathered the cell suspension by rinsing the vein with 20 mL of medium 131 (Gibco, Grand Island, NY, USA) enriched with 10% FBS. Following centrifugation 1200 rpm for 10 minutes, we cultivated the clustered cells in 25-mL laboratory flasks under humidified incubator conditions until individual colonies coalesced. Finally, we confirmed the endothelial phenotype of the cultivated cells by microscopy and immunohistochemistry for expression of von Willebrand factor (vWF).
Collagenase dispase
Manufactured by Roche
Sourced in Switzerland, United States, Germany, United Kingdom, Japan
Collagenase/dispase is a laboratory enzyme product used for the dissociation and disaggregation of various tissue types, including soft connective tissues. It functions by breaking down the extracellular matrix components, such as collagen and other glycoproteins, to facilitate the isolation and harvesting of individual cells from the tissue.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (53 mentions)
Dnase 1, by Merck Group (42 mentions)
Dnase, by Roche (18 mentions)
Dnase, by Merck Group (18 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Collagenase d, by Roche (11 mentions)
Dnase 1, by Worthington (9 mentions)
Facsaria 2, by BD (9 mentions)
Neurobasal medium, by Thermo Fisher Scientific (8 mentions)
Collagenase 2, by Worthington (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Percoll, by GE Healthcare (7 mentions)
Dynabead, by Thermo Fisher Scientific (7 mentions)
Lsrfortessa, by BD (7 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (7 mentions)
Ls column, by Miltenyi Biotec (6 mentions)
Egm 2mv, by Lonza (5 mentions)
Pharm lyse, by BD (5 mentions)
301 protocols using collagenase dispase
1
Isolation of Human Umbilical Vein Endothelial Cells
2
Isolation and Transcriptome Profiling of Leech Neurons
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Visually identified T, P, N and Retzius neurons were isolated individually from the central nervous system of adult H. verbana as described previously [75 (link), 76 (link)]. In brief, leeches were anesthetized by immersion in ice, and short chains of midbody segmental ganglia (with the exception of ganglia 5 and 6) were dissected in Leech Ringer’s solution [77 (link)] and pinned in a Sylgard-bottom dish. The ganglia were kept in L-15 culture medium (Gibco) supplemented with 6 mg/ml glucose, 0.1 mg/ml gentamycin (Sigma) and 2% heat-inactivated fetal calf serum (FCS, Microlab). The capsule of each ganglion was opened to expose cell somata and the ganglia chains were incubated for 1 h in 2 mg/ml collagenase/dispase (Boehringer-Mannheim). After the enzyme treatment, Retzius, T, P and N neurons were identified visually by their size and location in the ganglion. Individual neurons were removed from the ganglia by suction using a fire-polished glass pipette. Isolated neurons were rinsed several times in L-15 to sterilize them and remove debris. Three groups of ten leeches were used, yielding three biological replicates, of ~ 300 cells each, for each cell type. Finally, ganglia from which all the mechanosensory and Retzius neurons had been removed were pooled to create three biological replicates to create the “ganglion” transcriptome.
3
Isolation of Leech Nervous System Neurons
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Visually identified T, P, N and Retzius neurons were isolated individually from the central nervous system of adult H. verbana as described previously (Dietzel et al. 1986; de-Miguel and Vargas 1997) . In brief, leeches were anesthetized by immersion in ice, and short chains of midbody segmental ganglia (with the exception of ganglia 5 and 6) were dissected in Leech Ringer's solution (Muller and Scott 1981) and pinned in a Sylgard-bottom dish. The ganglia were kept in L-15 culture medium (Gibco) supplemented with 6 mg/ml glucose, 0.1 mg/ml gentamycin (Sigma) and 2% heatinactivated fetal calf serum (FCS, Microlab). The capsule of each ganglion was opened to expose cell somata and the ganglia chains were incubated for 1 hr in 2 mg/ml collagenase/dispase (Boehringer-Mannheim). After the enzyme treatment, Retzius, T, P and N neurons were identified visually by their size and location in the ganglion. Individual neurons were removed from the ganglia by use of a fire-polished glass pipette. Isolated neurons were rinsed several times in L-15 to sterilize them and remove debris. Three groups of ten leeches were used, yielding three biological replicates, of ~300 cells each, for each cell type. Finally, ganglia from which all the mechanosensory and Retzius neurons had been removed were pooled to create three biological replicates to create the "ganglion" transcriptome.
4
Lung Cell Isolation and Characterization
5
Lung Cell Isolation and Characterization
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Whole lung was dissociated in collagenase/dispase (Roche 10 269 63) and stained for the following markers of lung endothelium anti-mouse, CD31 PE (Biolegend); blood cells, anti-mouse CD45 PerCP-Cyanine5.5 and (Ebioscience) epithelium cells, CD326 (EpCAM) PECy7 (Biolegend). Staining was done with 0.1- 0.5 million cells with antibody dilutions of 1:100 for 20 minutes in the dark at room temperature. Cells were sorted on the BD Aria cytometer.
6
Isolation of Adipose Stromal Vascular Fraction
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Adipose tissue was dissected, rinsed in ice cold PBS, and minced with a razor blade. Fat was digested enzymatically with 3 mg/ml collagenase/dispase (Roche) at 37°C for 1 hour. Suspension was passed through a 70 μm strainer and centrifuged to pellet stromal vascular fraction. Cells were resuspended in PBS/0.04% BSA, counted, viability determined to be >90% before proceeding to flow cytometry analysis or scRNA-seq.
7
Lung Single-Cell Isolation Protocol
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After 4 °C preservation, lungs were enzymatically treated, and single‐cell suspensions were prepared as previously described with minor modifications 20 , 21 . In brief, the lungs were incubated in a 37 °C shaking incubator for 45 min in 10 mL of Dispase (2 U·mL−1; Roche Diagnostics, Indianapolis, IN, USA), 1 mL of DNase (0.1 mg·mL−1; Sigma‐Aldrich, St. Louis, MO, USA) and 1 mL of collagenase/Dispase (2 µg·mL−1; Roche Diagnostics). The lungs were then minced and incubated for 10 min. The cell suspension was filtered using a 40‐µm filter (BD Biosciences, San Jose, CA, USA).
8
Isolation of Brain Endothelial Cells
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For in vivo applications, the above procedure was modified to enrich for a single-cell suspension of brain endothelial cells without the need for culture. Cortices were homogenized and myelin was removed as above. Following the myelin separation, the pellets were combined and incubated in ACK buffer for 5 minutes followed by a 1-hour digestion in collagenase/dispase (Roche) and passed through a 70-µm strainer to produce a single-cell suspension. Samples were enriched for brain endothelial cells by depletion of remaining myelin debris with myelin separation beads and 3 rounds of positive selection with CD31 beads on a MulitMACS Cell24 Separator Plus. A fraction of the brain endothelial cell–enriched sample (15%) was assessed by flow cytometry. The remaining sample was pelleted for extraction of protein or RNA (see qRT-PCR). For protein extraction, pellets were lysed in 70 μL of RIPA buffer+1× HALT protease inhibitor cocktail, sonicated (20% amplification, 3 cycles), centrifuged (25 000g for 20 minutes at 4 °C), and supernatant was collected. Protein was not quantified as the BSA in the elution buffers masked the true protein levels of the samples. Experiments that typically require normalization to protein were instead normalized to actin values measured by Western blot.
9
Capillary Isolation from Mouse Brain and Lung
10
Skin and Lymph Node Tissue Dissociation
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