Once the bladder tumors became >7 mm in diameter, they were harvested for pathological evaluation, in vitro analysis and for establishing cell lines. Tumors were dissociated and digested with collagenase and dispase (Roche). The dissociated tumor cells were resuspended in growth media and plated to a plastic plate as previously described (26 (link)). Cell lines were passaged more than 10 times before use.
Collagenase and dispase
Manufactured by Roche
Sourced in United States, Germany
Collagenase and dispase are enzymes used in various laboratory applications, such as cell isolation and tissue dissociation. Collagenase breaks down collagen, a major component of the extracellular matrix, while dispase cleaves peptide bonds. Together, they facilitate the release and separation of cells from their surrounding matrix, enabling the isolation of specific cell types for further research or applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Red blood cell lysis buffer, by Roche (1 mentions)
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Ccl 229, by American Type Culture Collection (1 mentions)
Eagle s minimal essential medium, by Lonza (1 mentions)
Non essential amino acids (neaa), by Lonza (1 mentions)
Gelatin, by Merck Group (1 mentions)
Hepes, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Sodium bicarbonate, by Lonza (1 mentions)
Invitrogen countess 2, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Poly d lysine laminin coated glass coverslips, by BD (1 mentions)
7 protocols using collagenase and dispase
1
Establishing Bladder Tumor Cell Lines
2
Quantifying Tumor Protein Uptake and Immune Cell Profiling
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Tumors were injected with proteins labeled with Alexa FluorTM 647 (Invitrogen, A20006); for each protein, the degree-of-labeling was determined to be ~1.6 dye molecules per protein. Resected tumors were weighed, dissociated into small pieces, and incubated in RPMI-1640 containing 1 mg/ml collagenase and dispase (Roche) and 25 µg/ml Dnase I (Roche) for 30 min at 37 °C. Further mechanical dissociation through a 70 µm filter was used to generate a single cell suspension for staining. Staining using antibodies to CD11b (BV 421, clone M1/70, Biolegend 101251), Ly6-C (PE-Cy7, clone HK1.4, Biolegend 128017), and F4/80 (PerCP-Cy5.5, clone BM8, Biolegend 123127), CD45 (APC-Cy7, clone 30-F11, Biolegend 103115), CD3e (BUV 395, clone 145-2C11, BD Biosciences 563565), CD8a (BUV 737, clone 53-6.7, BD Biosciences 612759), NK1.1 (PerCP-Cy5.5, clone PK136, Biolegend 108727), CD4 (PE-Cy7, clone GK1.5, Biolegend 100421), CD25 (Alexa Fluor 700, clone PC61, Biolegend 102024), and FoxP3 (PE, clone 150D, Biolegend 320007) was performed at a 1:100 dilution. Viability was assessed by LIVE/DEAD Fixable Zombie Aqua (Biolegend 423101). Fluorescence quantitation of a tumor’s total Alexa Fluor 647 content was performed using Quantum™ Alexa Fluor® 647 MESF Beads (647, Bang Labs). Data was obtained using BD FACSDiva Software v6 and analyzed using FlowJo v10.7.1.
3
Isolation and Dissection of Embryonic Tissues
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Embryos were dissected in prewarmed Dulbecco’s PBS (DPBS) with 7% FCS to isolate yolk sac or FL. Single-cell suspensions were generated by incubating tissues in 10% collagenase and dispase (Roche) for 45 min at 37°C, washing, and manually dissociated by trituration. FLs were manually dissected into cross sections using sharpened tungsten needles. Peripheral blood was collected from individual embryos in prewarmed calcium- and magnesium-free DPBS with 7% FCS and EDTA as previously described (Potts et al., 2015 (link)).
4
Isolation and Characterization of Duck Aortic Endothelial Cells
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Primary duck aortic endothelial cells were isolated and characterized as previously described (25 (link)) and maintained in microvascular endothelial cell growth medium-2 (EGM-2MV; Lonza) on plates coated with 0.2% gelatin (Sigma-Aldrich) at 40°C in 5% CO2. Furin-deficient LoVo cells (CCL-229; ATCC) were maintained in Kaighn's modification of Ham's F-12 medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Sigma), 100 U/mL penicillin (Lonza), and 100 μg/mL streptomycin (Lonza), at 37°C in 5% CO2. Madin-Darby canine kidney cells (MDCK) were cultured in Eagle’s minimal essential medium (Lonza) supplemented with 10% FCS, 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM l- glutamine (Lonza), 1.5 mg/mL sodium bicarbonate (Lonza), 20 mM HEPES (Lonza), and nonessential amino acids (Lonza) at 37°C in 5% CO2. Duck lung homogenate (dLH) was obtained by digesting freshly isolated lungs of 21-day-old domestic duck embryonated eggs with collagenase and dispase (Roche) followed by treatment with a red blood cell lysis buffer (Roche) for 10 min at room temperature.
5
Isolation of Infected Tissue Cells
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Mock- and KSHV-infected tissues were harvested at 3 days and 6 days post infection. Tissue inserts were removed from the 6-well plate and placed into a new 6-well plate with enough PBS to coat the bottom. The inside of the insert was then washed with PBS to collect the supernatant. The tissues where then carefully separated from the insert membrane and chopped into small pieces in DMEM. The cells are then incubated for 40 minutes in 2 mL of 1 mg/mL collagenase and dispase (Roche Cat. 10269638001) in a shaking incubator set to 37°C and 250 rpm. The collagenase and dispase are then inactivated by adding 2 mL of DMEM and 10% FBS. The digested cells are then passed through a 0.45 um cell strainer and then washed with cold PBS with 0.04% BSA (ThermoFisher Cat. AM2618). The filtered cells are also passed through a flowmi cell strainer. Cells are counted with Invitrogen Countess II (ThermoFisher Cat. A27977).
6
Culturing TG Neurons for Calcium Imaging
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Mouse TG neurons were cultured for calcium imaging experiments. Naïve male and female mice were injected with WGA as described above, bilaterally in the tongue and 2 days later, TGs were dissected for neuronal cultures as described previously125 (link). Tissues were washed in HBSS and then dissociated with 1 mg/ml of collagenase and dispase (Roche, Indianapolis, IN, USA) for 45 min at 37 degrees. Following washing out the enzymes, cultures were plated on poly-D-lysine/laminin coated glass coverslips (BD Biosciences, San Jose, CA, USA) and grown overnight in either DMEM media supplemented with glutamine, penicillin/streptomycin and 2% Fetal bovine serum (for vehicle group) or the same media with 100 ng/ml recombinant colony-stimulating factor 1 (Csf1, Cell Signaling, Boston, MA, USA). Csf1 stock was diluted in saline.
7
Isolation of Neonatal Mouse Cardiomyocytes
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All animal experimental procedures were approved by the Animal Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command of PLA. Primary neonatal mouse cardiomyocytes (NMCMs) were isolated from newborn C57BL/6 mice, as previously described (19) . In brief, the mouse heart was isolated and cut into small pieces. Then, the tissue pieces were digested with collagenase and dispase (Roche, Penzberg, Upper Bavaria, Germany), leading to the cells into suspension. The cell suspension was added to a cell strainer to remove larger tissue fragments and then preplated for 2 h to remove fibroblasts and endothelial cells. After incubation, nonadherent cardiomyocytes were centrifuged and plated into collagen-coated cell culture dishes with DMEM medium containing 10% fetal bovine serum (FBS; HyClone, Salt Lake City, Utah) in a humidified atmosphere with 5% CO 2 at 37°C. Cells of passages 3 to 4 were used in this study.
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