We cloned and expressed canine-ized cytokines IL-2 and IL-12 fused to the collagen-binding domain LAIR1 as detailed in the Supplementary Methods S1 (sequences in Supplementary Table S1 ). To assess IL-2 bioactivity, 10,000 CTLL-2 cells (ATCC Cat# TIB-214, RRID:CVCL_0227) were plated per well in incomplete T-cell media (RPMI 1640, 10% fetal bovine serum, 2mM L-glutamine, 1mM sodium pyruvate) and incubated for 48h at 37°C with dilutions of the IL-2 fusion protein. After incubation, cell viability was assessed using the CellTiter-Glo 2.0 assay (Promega). The HEK Blue IL-12 (Invivogen; RRID:CVCL_UF31) assay was performed according to vendor instructions using dilutions of the collagen-binding IL-12 cytokine. Collagen-binding of both cytokines was evaluated through ELISA with rat collagen I-coated plates and an anti-hexahistidine detection antibody (Abcam Cat# ab1269, RRID:AB_299333) as previously reported(16 (link)). Low endotoxin levels (<5 EU/kg/dose) were confirmed for each cytokine batch using the Endosafe Nexgen-PTS system (Charles River Labs) with typical values <1 EU/mg protein.
Tib 214
Manufactured by American Type Culture Collection
The TIB-214 is a piece of laboratory equipment designed for the cultivation and storage of cell cultures. It provides a controlled environment for the growth and maintenance of various cell lines, ensuring optimal temperature, humidity, and gas conditions. The core function of the TIB-214 is to facilitate the culturing and preservation of cells used in scientific research and applications.
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2 protocols using tib 214
1
Evaluating Engineered Canine Cytokines
2
T Cell Proliferation Assay for Fusion Proteins
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The biological activity of the fusion proteins was evaluated by their capability to induce the proliferation of murine cytotoxic T lymphocytes, CTLL2 (ATCC, TIB-214™). In 96-well plates, cells (25,000–50,000 per well) were seeded in culture medium supplemented with serial dilutions of the fusion proteins. After incubation at 37 °C for 72 h, cell proliferation was measured with Cell Titer Aqueous One Solution (Promega, Madison, WI, USA). Results were represented as the percentage of cell viability compared with unstimulated cells.
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