A single-cell suspension was prepared from mouse LNs and spleens (both male and female mice, 6 to 8 weeks old). For the lung tissues, lungs were cut into small pieces and then digested for 20 min using digestion buffer containing collagenase IV, hyaluronidase, and deoxyribonuclease I. For γδT17 cell polarization, CD27− γδ T cells from LNs were cultured with IL-1β (10 ng/ml) and IL-23 (10 ng/ml) for 7 days in wells precoated with anti–γδ TCR monoclonal antibody (mAb) (clone: UC-7). For γδT1 cell polarization, sorted CD27+ γδ T cells from pooled LNs and spleens were cultured with IL-2 (10 ng/ml) and IL-7 (10 ng/ml) for 7 days in wells precoated with anti-CD3 mAb (clone: 145-2C11). In some experiments, dimethyl 2-oxoglutarate (α-KG, Sigma-Aldrich), N-acetyl-l -cysteine (Sigma-Aldrich) were added during the culture. γδT17 and γδT1 cells were further sorted from cultures based on CD27 expression. Cells were sorted using FACSAria III cell sorter (BD Biosciences). The detailed commercial Ab information is listed in table S1.
Dimethyl 2 oxoglutarate α kg
Manufactured by Merck Group
Sourced in United States
Dimethyl 2-oxoglutarate (α-KG) is a chemical compound used as a laboratory reagent. It has the molecular formula C₆H₈O₅. The compound is a diester of 2-oxoglutaric acid (also known as α-ketoglutaric acid).
Automatically generated - may contain errors
Lab products found in correlation
N acetyl l cysteine, by Merck Group (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Aminooxyacetate, by Merck Group (1 mentions)
Torin1, by Bio-Techne (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Antibodies against p aktser473, by Cell Signaling Technology (1 mentions)
Chop d46f1, by Cell Signaling Technology (1 mentions)
Fitc conjugated secondary antibody, by Agilent Technologies (1 mentions)
Epigallocatechin gallate (egcg), by Selleck Chemicals (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
2 protocols using dimethyl 2 oxoglutarate α kg
1
Polarization of Mouse γδ T Cells
2
Recombinant TRAIL Protein Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Soluble human His-tagged recombinant TRAIL was generated in our laboratory as described44 (link). Anti-caspase 8 antibody was generously provided by Dr. Gerald Cohen (Leicester University, UK). Antibodies against p-AKTSer473, AKT, p-P70S6K, P70S6K, PERK (C33E10), eIF2α (D7D3), p-eIF2α and CHOP (D46F1) were obtained from Cell Signaling Technology (CA, USA). Antibodies against ATF4, GAPDH, and GCN2 were obtained from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-TRAIL-R2 antibody was purchased from R&D Systems (Minneapolis, USA). Anti-FLIP (7F10) antibody was from Enzo Life Sciencies (Farmingdale, NY, USA). p4E-BP1 and 4E-BP1 antibodies were from Upstate Millipore (NY, USA). FITC-conjugated secondary antibodies were from DAKO (Cambridge, UK). Anti-TRAIL-R1-PE and anti-TRAIL-R2-PE monoclonal antibodies for surface receptor analysis were from Biolegend (San Diego, CA, USA). Aminooxyacetate, non-essential amino-acid solution (100×), cycloheximide, dimethyl 2-oxoglutarate (α-KG), and l -Asparaginase from E.Coli were from Sigma-Aldrich (St. Louis, MO, USA). Torin1 was purchased from TOCRIS Bioscience (Bristol, UK). Rapamycin was purchased from LC laboratories (Woburn, MA, USA). Epigallocatechin Gallate (EGCG) was purchased from Selleck Chemicals (Houston, Texas, USA).
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