NK cell and CD8 T cell enrichment were performed using the CD56+ CD16+ NK and CD8+ T Cell Isolation Kit (Miltenyi Biotec), respectively. After magnetic cell separation, NK and CD8+ T cells were seeded in 6-plate wells cells at the density of 106 cells/well and treated with 25 μM of TMZ or vehicle (DMSO), for 10 and 30 min, were washed with cold PBS and lysed in a buffer supplemented with protease and phosphatase inhibitors. Membranes with transferred proteins were incubated with the primary antibody anti-pNRF2 (phosphoSer40, 1:5000, Abcam), anti-NRF2 (1:1000, Abcam) or anti-vinculin (1:10,000). The primary antibody incubation was followed by incubation with peroxidase conjugated to the secondary antibody (anti-rabbit, 1:10,000). A chemiluminescence reaction using the ECL Plus kit (GE Healthcare, Chicago, IL, USA) was detected using G: BOX iChemi system (Syngene, Cambridge, UK).
G box ichemi system
Manufactured by Syngene
Sourced in United States, United Kingdom
The G:BOX iChemi system is a versatile imaging system designed for a range of applications in life science research. It provides high-quality imaging capabilities for various assays and sample types. The system utilizes a sensitive CCD camera and a selection of illumination sources to capture images of chemiluminescent, fluorescent, and colorimetric samples. Its core function is to enable researchers to visualize and analyze a variety of biological samples with precision and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus kit, by GE Healthcare (2 mentions)
Anti nrf2, by Abcam (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Swine anti rabbit hrp conjugated antibody, by Agilent Technologies (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
G box chemi system, by Syngene (1 mentions)
Mouse cytokine array panel a kit, by R&D Systems (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
3 protocols using g box ichemi system
1
Investigating TMZ-Induced Stress Response in NK and CD8+ T Cells
2
Quantifying α-Tubulin Acetylation
3
Western Blot and Cytokine Analysis
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Briefly, cells were washed with cold PBS and lysed in a buffer supplemented with protease and phosphatase inhibitors. Membranes with transferred proteins were incubated with the primary antibody anti-pAKT (Ser473) (1:1000, Cell Signaling) or anti-vinculin (1:10000). The primary antibody incubation was followed by incubation with peroxidase conjugated to the secondary antibody (anti-rat, 1:10000). A chemiluminescence reaction using the ECL Plus kit (GE Healthcare) was detected using G:BOX iChemi system (Syngene). Tumor relative levels of cytokines and chemokines were measured using the Mouse Cytokine Array Panel A kit (R&D Systems) following the manufacturer's instructions. Images of the blots were acquired with G:BOX Chemi system (Syngene) and quantitative analyses were performed using ImageJ software. The 40 cytokines and chemokines of interest were normalized to the corresponding positive controls.
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