Peripheral leukocytes were isolated by Ficoll density gradient centrifugation. TILs were obtained from fresh tissue samples, as described previously (12). In brief, the tumor masses were minced, and digested with collagenase (type I and type IV, 0.05 mg/mL, Sigma) and DNase I (0.05 mg/mL, Roche) solution at 37°C for 1 hour. The cell suspension was filtered through a cell mesh and resuspended in RPMI1640 medium for further analysis. Peripheral leukocytes were isolated by Ficoll density gradient centrifugation. Thereafter, the mononuclear cells were washed and resuspended in RPMI1640 medium supplemented with 10% FBS. CD14+ monocytes/macrophages and CD3+ T lymphocytes were isolated using magnetic beads (130–050–201/130–095–130, Miltenyi Biotec) for use in subsequent ex vivo or in vitro experiments.
Collagenase type 1 and type 4
Manufactured by Merck Group
Sourced in United States
Collagenase is an enzyme that is used to break down collagen, a structural protein found in various tissues. Merck Group offers Collagenase Type I and Type IV. Type I Collagenase is effective in digesting native collagen from a variety of sources, while Type IV Collagenase is primarily used to digest basement membrane collagen.
Automatically generated - may contain errors
2 protocols using collagenase type 1 and type 4
1
Isolation of Peripheral Leukocytes and TILs
2
Isolation and Culture of Crayfish HPT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HPT cells were isolated and cultured as previously described [11, 17] . Briefly, after dissection of the crayfish, HPT was dissected and incubated for 20 min in 800 µl of 0.1% collagenase type I and type IV (Sigma-Aldrich, USA) in crayfish phosphate-buffered saline (CPBS; 10 mM Na 2 HPO 4 , 10 mM KH 2 PO 4 , 150 mM NaCl, 10 mM CaCl 2 and 10 mM MnCl 2 , pH 6.8) at room temperature. Next, the collagenase solution was removed after being centrifuged at 800 x g for 5 min. The resulting cell pellet was washed twice with 1 ml of CPBS and then suspended in L-15 medium (Sigma-Aldrich, USA) supplemented with 1 mM phenylthiourea, 60 mg/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin (Sigma-Aldrich, USA), and 2 mM L-glutamine. The cells were cultured in 96-well plates at a density of 5 x 10 4 cells/well at 16 °C. One third of the medium was changed every second day.
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!