TILs were isolated from the tumors according to previous report [20 (link)]. Briefly, small pieces of solid tumors tissue was digested with an enzyme cocktail containing 2% fetal bovine serum, 0.5 mg/ml collagenase A (Roche), 0.2 mg/ml hyaluronidase, type V (Sigma) and 0.02 mg/ml DNase I (Sigma) per 0.25 g of tumor tissue. The cell suspensions were filtered through a cell strainer (70 μm, Becton Dickinson, CA, USA), and then, they were washed with 2% FBS in RPMI 1640. After lysed red blood cells, the cell clumps were removed by 40%/70% Percoll gradient centrifugation and centrifuged at 400 g. The nonadherent lymphocytes were harvested and CD4+ lymphocytes were isolated by Dynabeads M-450 CD4 according to the manufacturer’s instruction. These CD4+ lymphocytes were further isolated into CD25+ and CD25- lymphocytes by Dynabeads (Dynal Biotech ASA, Oslo, Norway). CD4 + CD25+ T cells and/or CD4 + CD25- T cells were added different concentrations of GLPS, and cultured for 72 h, at 37°C and 5% CO2.
Type 5
Manufactured by Merck Group
Sourced in Germany
The Type V lab equipment from Merck Group is designed for versatile laboratory applications. It provides core functionality for various experimental and analytical processes, while maintaining a concise and unbiased description of its primary functions.
Automatically generated - may contain errors
3 protocols using type 5
1
Isolation and Stimulation of TILs
2
Intestinal Permeability Measurement
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Intestinal paracellular permeability across cell monolayers was determined by measuring the flux of horseradish peroxidase (HRP; type V; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). HRP (3.4×10−6 mol/l) was added to the medium in the apical chamber of Transwell chambers. After pre-treatment with LPS for 30 min and exposure to heat stress for 1 h, samples were carefully taken from basolateral chambers and assayed for HRP by TMB HRP Color Development Solution for ELISA (Beyotime, China) 6 h later. Enzyme activity was determined from the rate of increase in optical density at a wavelength of 370 nm by an automatic microplate reader (SpectraMax® M5; Molecular Devices, LLC, Sunnyvale, CA, USA).
3
Islet Isolation and Transplantation Protocol
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