HEK293 cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin. All tumor samples were deidentified prior to use, and IRB approval from Yale University, along with informed consent from all included study participants, was obtained. The tumor sample was washed in 1X RPMI, chopped into small pieces (<1 mm), and the fragments were digested with a mixture of collagenase type I and IV (0.5 mg/mL in PBS; Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37 °C. A single-cell suspension was prepared straining the cells through a 100-μm mesh cell strainer and spun at 1,000 g for 5 min. The cell pellet was resuspended in DMEM/F12 supplemented with 20% fetal bovine serum (Life Technologies), in a humidified atmosphere of 5% CO2/air. Cells were split at no more than 1:3 per passage and used up to passage 7.
Collagenase type 1 and 4
Manufactured by Merck Group
Sourced in United States
Collagenase type I and IV are enzymes used in cell and tissue culture applications. They function to break down collagen, the primary structural protein in connective tissues. These collagenases are commonly used to dissociate and isolate cells from various tissue types.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using collagenase type 1 and 4
1
Isolation and culture of primary tumor cells
2
Isolation and Culture of Canine Adipose-Derived Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adipose tissue was harvested from purebred healthy dogs (n = 3) and was collected under general anaesthesia, maintaining the principles of sterility and asepsis, from the subcutaneous tissue in the scapular area. Adipose tissue was collected from identical donors as for bone marrow (Section 2.1 ). We isolated 5–7 g of adipose tissue from each donor. The tissue was then washed with phosphate-buffered saline (PBS; Biowest) containing 2% ATB + ATM, then mechanically dissociated and enzymatically digested with 0.05% collagenase type I and IV (Sigma, Burlington, MA, USA) at 37 °C for 1 h. At the end of the incubation period, the digested tissue was filtered (through a 100 μm cell strainer) to remove tissue fragments, centrifuged at 400× g for 10 min, and the obtained stromal vascular fraction (SVF) pellet was resuspended in culture medium consisting of DMEM-F12 containing 10% FBS and 2% ATB + ATM and plated in a T25 tissue culture flask at a concentration of 106 cells/flask and incubated in cultivation medium (DMEM-F12 + 10% FBS + 2% ATB + ATM) at 37 °C and 5% CO2. After 48 h, non-adherent cells were removed and, subsequently, the medium was changed twice a week.
3
Canine Adipose Tissue Isolation and Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adipose tissue (AT) was harvested from purebred healthy dogs (n = 3): Donor 1-male, German Shepherd, 35 kg, 3 years old. Donor 2-female, Cane Corso, 70 kg, 4 years old. Donor 3-male, German Shorthaired Pointer, 32 kg. Before adipose tissue collection, all donors were examined (clinical examination, biochemical, and hematological parameters were evaluated). Samples of adipose tissue (5-7 g) were collected during surgical procedures under general anesthesia from the subcutaneous tissue in the scapular area. The tissue was then washed with phosphate-buffered saline (PBS; Biowest, Nuaillé, France) containing 2% ATB + ATM, then mechanically dissociated and enzymatically digested with 0.05% collagenase type I and IV (Sigma, Burlington, MA, USA) at 37 • C for 1 h. At the end of the incubation period, the digested tissue was filtered (through a 100 µm cell strainer) to remove tissue fragments, centrifuged at 400× g for 10 min, and the obtained stromal vascular fraction (SVF) pellet was resuspended in culture medium consisting of DMEM-F12 containing 10% FBS and 2% ATB + ATM and plated in a T25 tissue culture flask at a concentration of 106 cells/flask and incubated in cultivation medium (DMEM-F12 + 10% FBS + 2% ATB + ATM) at 37 • C and 5% CO 2 . After 48 h, non-adherent cells were removed, and subsequently, the medium was changed twice a week.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!