To obtain single melanoma cell suspensions using sterile scissors and tweezers, we first removed adipose or and necrotic tissue before cutting melanoma tumour tissues into fragments (with volumes less than 1 mm3) on sterile cell culture dishes. Tissue digestion was required for our experiment, but acral melanoma tumour tissues are rigid and not easily digested. By exploring different procedures, we found that a combination of 0.2% collagenase IV (Sigma, Cat# V900893) and 0.1% Dispase II enzymes (Sigma, Cat# D4693) was optimal for the digestion of acral melanoma tumour tissues. The tissues were incubated in a solution containing collagenase IV and Dispase II enzymes for approximately 30 min at 37°C. Then, the cell suspensions were filtered through a sterile cell strainer and centrifuged at 1000 rpm for 10 min at room temperature. Then, the resulting pellets were collected and resuspended in 2 ml of complete Opti‐MEM each, along with 100 U/ml penicillin, 100 U/ml streptomycin and 1.25 U/ml nystatin, in a 3.5 cm dish. These dishes were then incubated at 37°C in 5% CO2. After 2 days, the medium was removed and the cells were washed with PBS. The medium was refreshed every 2 days until the primary cells reached confluence.
Dispase 2 enzyme
Manufactured by Merck Group
Sourced in United States, Singapore, China
Dispase II is a protease enzyme derived from Bacillus polymyxa. It is used for the dissociation and disaggregation of a variety of cell and tissue types, including epithelial, endothelial, and connective tissues.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase 4, by Merck Group (2 mentions)
Collagenase a, by Merck Group (2 mentions)
Tms ab2 c, by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Dmem f12 medium, by Merck Group (1 mentions)
Leibovitz s l 15 medium, by Merck Group (1 mentions)
Pdms sylgard 184, by Dow (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
Accutase solution, by PromoCell (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
6 protocols using dispase 2 enzyme
1
Melanoma Cell Isolation Protocol
2
Isolation and Characterization of Primary Cutaneous Squamous Cell Carcinoma Cells
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The primary SCC tumor tissues were obtained from patients in the Department of Dermatology at Xiangya Hospital, with informed consent from the patient. All experiment protocols were reviewed and approved by the Clinical Medical Ethics Committee, Xiangya Hospital, Central South University (202305092). First, the adipose and necrotic tissue of the tumor was removed and then the tumor was cut into fragments (< 1 mm3) in sterile tubes. Then, 1.5 ml of PBS containing 0.2% collagenase IV (Sigma, USA) and 0.1% Dispase II enzymes (Sigma, USA) was added and incubated at 37 °C for 1 h. Subsequently, the cell supernatant was filtered through 100-mesh and 40-mesh cell screens in turn. The cell suspension was centrifuged at 400 g for 8 min at 4 °C and washed twice with D-Hank’s solution. Finally, the cell precipitation was collected and resuspended in 2 ml of complete Opti-MEM medium in a 6-well plate. The cells were incubated at 37 °C in 5% CO2 and the medium was refreshed every 2 d until the primary cells attach to the plate (around 7 d) [34 ]. Then, the immunofluorescence analysis on SERPINB3 (a biomarker of SCC) was determined for the attached primary cells, and an in vitro cell uptake experiment was carried out as above mentioned above.
3
Isolation of Mouse Corneal Stromal Cells
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The removed mouse eyeballs were washed with sterile PBS containing penicillin and streptomycin (100 U/mL, TMS-AB2-C; Merck Millipore, Boston, MA, USA) three times. Each eyeball was digested for 12 hours at 4°C with 150 µL dispaseII enzyme (15 mg/mL; Sigma-Aldrich). Next, the corneal tissues were cut into small fragments after the removal of loose corneal epithelium. Furthermore, the tissues were digested with collagenase A (10 mg/mL; Sigma-Aldrich) at 37°C for 1 hour. Then, corneal stromal cells were suspended in a 25-cm2 culture flask with DF12 medium (DMEM F-12; Sigma-Aldrich). Adherent cells were observed after 10 to 12 hours at 37°C.
4
Mouse Corneal Stromal Cell Isolation
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Mouse eyeballs were extracted and digested with 0.2 mL dispase II enzyme (Sigma–Aldrich) at 4°C overnight. After the loose corneal epithelium and endothelium were removed, the tissues were gently minced and homogenized with 0.5 mL collagenase A (Sigma–Aldrich) at 37°C for 1 hour. The digestion was terminated with DMEM F-12 medium (Sigma–Aldrich) containing 10% fetal bovine serum. The cell suspension was inoculated in a cell culture flask. Eventually, the corneal stromal cells were adhered to the culture flask wall and passaged every 3 to 5 days.
5
Porcine Esophagus Tissue Engineering
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PDMS (SYLGARD 184) was purchased from Dow Corning and 1 mm thick PMMA sheets were bought from a local workshop. Small assembly components and tools including ring magnets, stainless steel screws, epoxy adhesives, surgical scissors and scalpel were purchased from RS Components. Fetal Bovine Serum (FBS) and phosphate-buffered saline (PBS) solutions were purchased online from ThermoFisher Scientific Singapore. All other bioreagents/chemicals such as Keratinocyte Growth Medium (KGM), Leibovitz’s L-15 medium, fluorescein isothiocyanate–dextran (FITC-Dextran), sodium alginate, acetic acid, dispase II enzyme, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) and antibiotic antimycotic (100×) solutions were purchased from Sigma Aldrich Singapore (Sigma-Aldrich Pte Ltd, 2 Science Park Drive, #05-01/12 Ascent Building, Singapore 118222). Fresh porcine esophagus was harvested from pigs euthanized in Singapore General Hospital (SGH) and delivered to the lab for subsequent processing within three hours. During transportation, the harvested esophagus was kept in ice-cold Leibovitz’s L-15 medium.
6
Melanocyte Cultivation and Protein Extraction
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M2 melanocyte medium, human melanocyte growth stimulating factors, and Accutase Solution were purchased from Promocell (PromoCell GmbH, Jiangsu Punuo Biotechnology Co., Ltd, Wuxi City, China), while dispase II enzyme and Coomassie brilliant blue were obtained from Sigma-Aldrich (St. Louis, MO, USA), 0.25% trypsin-EDTA was purchased from Invitrogen, fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG was obtained from Euroimmun, and the fluorescence microscope was purchased from Olympus Corporation (Tokyo, Japan). The membrane protein, nucleoprotein, and cytoplasmic protein extraction kits were purchased from KeyGEN Biotech, while the bicinchoninic (BCA) protein quantification kit and the horseradish peroxidase (HRP)-DAB chromogenic substrate kit were obtained from Tiangen Biotech. The HRP-labeled rabbit anti-human IgG antibody was a product of Bioss, and the human monoclonal antibody ELISA Kit and Human HSP70 ELISA Kit were provided by KainuoBio Corporation. The human hepatitis B surface antigen (HBsAg) ELISA kit was purchased from Wantai (Beijing, China), while the enzyme-labeling measuring instrument was a product of Bio-Rad (Hercules, CA, USA).
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