Filtration:Article Title: 4-Nitro-3-phenylphenol has both androgenic and anti-androgenic-like effects in rats
Article Snippet: .. After that, the testes were dissociated by incubation in M199 medium (Gibco®) containing 0.71 g/l sodium bicarbonate, 2.21 g/l HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1% BSA, and 25 mg/l soybean trypsin inhibitor (STI), pH 7.4, with 0.25 mg/ml of collagenase (Worthington Biochemical, Freehold, NJ, USA) and then horizontal shaking (90 cycles/min) at 34 C for approximately 10–20 min. After dissociation, the seminiferous tubules were removed by filtration through 100 µm nylon mesh. ..
Avidin-Biotin Assay:Article Title: Isolation of carbohydrate-specific CD4+ T cell clones from mice after stimulation by two model glycoconjugate vaccines
Article Snippet: .. Penicillin/streptomycin (Cellgro, cat. no. 30002201) Gentamicin (Gibco, cat. no. 15750-060) Sodium bicarbonate (Worthington, cat. no. P8N10891) ACK lysing buffer (Invitrogen, cat. no. A1049201) T cell culture supplement without ConA (TCCS, IL-2 culture supplement; BD Biosciences, cat. no. 354116) Multiscreen HA 96-well ELISpot plate (Millipore, cat. no. MAHAS4510) Anti-mouse IL-2 capture antibody, clone JES6-1A12 (Biolegend, cat. no. 503704) Biotin-labeled anti-mouse IL-2 antibody, clone JES6-5H4 (Biolegend, cat. no. 503804) Anti-mouse IL-4 capture antibody, clone 11B11 (Biolegend, cat. no. 504115) Biotin-labeled anti-mouse IL-4 antibody BVD6-24G2 (Biolegend, cat. no. 504202) Avidin-HRP (Biolegend, cat. no. 405103) AEC ELISpot substrate kit (BD Bioscience, cat. no. 551951) TruStain Fcx anti-mouse CD16/32 antibody (Biolegend, cat. no. 101320) FITC-labeled anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100306) APC anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100312) FITC anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100406) PerCp anti-mouse CD8 antibody, clone 53.67 (Biolegend, cat. no. 100732) PE anti-mouse TCRβ chain antibody, clone H57-597 (Biolegend, cat. no. 109208) Allophycocyanin-labeled anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100412) LEAF purified anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100435) LEAF purified anti-mouse CD8a antibody, clone 53-6.7 (Biolegend, cat. no. 100735) Ethanol Trypan blue stain DMSO Tween-20 .. Vial, 1 dram pH meter Magnetic stir bars Magnetic stir plate PD-10 columns (GE Healthcare, cat. no. 17-0851-01) Sterile syringe-driven filter unit (0.22 µm) Freeze dryer (Freezemobile 12SL) Ultraviolet spectrophotometer (Beckman, cat. no. DU 640) BioLogic HR FPLC system (Bio-Rad) Superdex 200 HiLoad 16/60 prep grade column (GE Healthcare) Dialysis membrane (molecular weight cutoff (MWCO): 12,000–14,000 Da; Spectrum Labs) 96-Well plate, flat-bottomed PowerWave HT microplate reader (Bio-Tek) Criterion TGX any kD stain-free gel (Bio-Rad) Mini-PROTEAN Tetra Cell (Bio-Rad) ChemiDoc MP imager (Bio-Rad)
Cell Culture:Article Title: Isolation of carbohydrate-specific CD4+ T cell clones from mice after stimulation by two model glycoconjugate vaccines
Article Snippet: .. Penicillin/streptomycin (Cellgro, cat. no. 30002201) Gentamicin (Gibco, cat. no. 15750-060) Sodium bicarbonate (Worthington, cat. no. P8N10891) ACK lysing buffer (Invitrogen, cat. no. A1049201) T cell culture supplement without ConA (TCCS, IL-2 culture supplement; BD Biosciences, cat. no. 354116) Multiscreen HA 96-well ELISpot plate (Millipore, cat. no. MAHAS4510) Anti-mouse IL-2 capture antibody, clone JES6-1A12 (Biolegend, cat. no. 503704) Biotin-labeled anti-mouse IL-2 antibody, clone JES6-5H4 (Biolegend, cat. no. 503804) Anti-mouse IL-4 capture antibody, clone 11B11 (Biolegend, cat. no. 504115) Biotin-labeled anti-mouse IL-4 antibody BVD6-24G2 (Biolegend, cat. no. 504202) Avidin-HRP (Biolegend, cat. no. 405103) AEC ELISpot substrate kit (BD Bioscience, cat. no. 551951) TruStain Fcx anti-mouse CD16/32 antibody (Biolegend, cat. no. 101320) FITC-labeled anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100306) APC anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100312) FITC anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100406) PerCp anti-mouse CD8 antibody, clone 53.67 (Biolegend, cat. no. 100732) PE anti-mouse TCRβ chain antibody, clone H57-597 (Biolegend, cat. no. 109208) Allophycocyanin-labeled anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100412) LEAF purified anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100435) LEAF purified anti-mouse CD8a antibody, clone 53-6.7 (Biolegend, cat. no. 100735) Ethanol Trypan blue stain DMSO Tween-20 .. Vial, 1 dram pH meter Magnetic stir bars Magnetic stir plate PD-10 columns (GE Healthcare, cat. no. 17-0851-01) Sterile syringe-driven filter unit (0.22 µm) Freeze dryer (Freezemobile 12SL) Ultraviolet spectrophotometer (Beckman, cat. no. DU 640) BioLogic HR FPLC system (Bio-Rad) Superdex 200 HiLoad 16/60 prep grade column (GE Healthcare) Dialysis membrane (molecular weight cutoff (MWCO): 12,000–14,000 Da; Spectrum Labs) 96-Well plate, flat-bottomed PowerWave HT microplate reader (Bio-Tek) Criterion TGX any kD stain-free gel (Bio-Rad) Mini-PROTEAN Tetra Cell (Bio-Rad) ChemiDoc MP imager (Bio-Rad)
Mouse Assay:Article Title: Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production
Article Snippet: .. Whole lungs and the distal 15 cm of the small intestine were removed from selected mice and incubated in digest media for 45 min followed by the addition of EDTA (2 mM final concentration) for 15 min. Digest medium consisted of Dulbecco’s modified Eagle’s media supplemented with 5% fetal calf serum, sodium bicarbonate, penicillin/streptokinase, 10 mM Hepes, 250 U/mL collagenase I (Worthington Biochemical), 50 U/mL DNase I (Worthington Biochemical), and 0.01% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) with or without 0.5 mg Brefeldin A (Sigma-Aldrich) in DMSO. .. The samples were filtered and the single cell suspension was treated with Red Blood Cell Lysing Buffer (Sigma-Aldrich) with or without Brefeldin A. APC-, FITC-, or PE-labeled antibodies against murine Mac-3/CD107b (clone M3/84), NK1.1 (clone PK 136), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3ε (clone 145-2C11), CD11b (clone M1/70), CD11c (clone N418), FcεRI (clone MAR-1), anti-mouse Lineage Cocktail (catalog #133305), and isotype controls IgGs (rat and Armenian hamster) were obtained from BioLegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), and/or BD Pharmingen (San Jose, CA, USA).
Concentration Assay:Article Title: Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production
Article Snippet: .. Whole lungs and the distal 15 cm of the small intestine were removed from selected mice and incubated in digest media for 45 min followed by the addition of EDTA (2 mM final concentration) for 15 min. Digest medium consisted of Dulbecco’s modified Eagle’s media supplemented with 5% fetal calf serum, sodium bicarbonate, penicillin/streptokinase, 10 mM Hepes, 250 U/mL collagenase I (Worthington Biochemical), 50 U/mL DNase I (Worthington Biochemical), and 0.01% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) with or without 0.5 mg Brefeldin A (Sigma-Aldrich) in DMSO. .. The samples were filtered and the single cell suspension was treated with Red Blood Cell Lysing Buffer (Sigma-Aldrich) with or without Brefeldin A. APC-, FITC-, or PE-labeled antibodies against murine Mac-3/CD107b (clone M3/84), NK1.1 (clone PK 136), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3ε (clone 145-2C11), CD11b (clone M1/70), CD11c (clone N418), FcεRI (clone MAR-1), anti-mouse Lineage Cocktail (catalog #133305), and isotype controls IgGs (rat and Armenian hamster) were obtained from BioLegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), and/or BD Pharmingen (San Jose, CA, USA).
Incubation:Article Title: 4-Nitro-3-phenylphenol has both androgenic and anti-androgenic-like effects in rats
Article Snippet: .. After that, the testes were dissociated by incubation in M199 medium (Gibco®) containing 0.71 g/l sodium bicarbonate, 2.21 g/l HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1% BSA, and 25 mg/l soybean trypsin inhibitor (STI), pH 7.4, with 0.25 mg/ml of collagenase (Worthington Biochemical, Freehold, NJ, USA) and then horizontal shaking (90 cycles/min) at 34 C for approximately 10–20 min. After dissociation, the seminiferous tubules were removed by filtration through 100 µm nylon mesh. ..
Article Title: Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production
Article Snippet: .. Whole lungs and the distal 15 cm of the small intestine were removed from selected mice and incubated in digest media for 45 min followed by the addition of EDTA (2 mM final concentration) for 15 min. Digest medium consisted of Dulbecco’s modified Eagle’s media supplemented with 5% fetal calf serum, sodium bicarbonate, penicillin/streptokinase, 10 mM Hepes, 250 U/mL collagenase I (Worthington Biochemical), 50 U/mL DNase I (Worthington Biochemical), and 0.01% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) with or without 0.5 mg Brefeldin A (Sigma-Aldrich) in DMSO. .. The samples were filtered and the single cell suspension was treated with Red Blood Cell Lysing Buffer (Sigma-Aldrich) with or without Brefeldin A. APC-, FITC-, or PE-labeled antibodies against murine Mac-3/CD107b (clone M3/84), NK1.1 (clone PK 136), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3ε (clone 145-2C11), CD11b (clone M1/70), CD11c (clone N418), FcεRI (clone MAR-1), anti-mouse Lineage Cocktail (catalog #133305), and isotype controls IgGs (rat and Armenian hamster) were obtained from BioLegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), and/or BD Pharmingen (San Jose, CA, USA).
Article Title: Serum-induced changes in the physiology of mammalian retinal glial cells: role of lysophosphatidic acid
Article Snippet: .. In brief, approximately 0.5 cm × 0.5 cm pieces of retina were incubated in Earle's balanced salt solution supplemented with 0.5 mM EDTA, 1.5 mM CaCl2 , 1 mM MgSO4 , 20 mM glucose, 26 mM sodium bicarbonate, 15 u papain (Worthington Biochemicals Co.), 0.04% DNase, 2 mM cysteine and 12% chicken serum for 40 min at 30°C, whilst 95% oxygen-5% CO2 was bubbled through to maintain pH and oxygenation. .. The piece of retina was then washed with the appropriate bathing solution, drawn up into a glass pipette and gently ejected back into a microcentrifuge tube.
other:Article Title: A population of central complex neurons promoting airflow-guided orientation in Drosophila
Article Snippet: Briefly, Drosophila saline contained 103 mM sodium chloride, 3 mM potassium chloride, 5 mM TES, 8 mM trehalose dihydrate, 10 mM glucose, 26 mM sodium bicarbonate, 1 mM sodium phosphate monohydrate, 1.5 mM calcium chloride dihydrate, and 4 mM magnesium chloride hexahydrate.
Article Title: Effect of Long-Term Osmotic Loading Culture on Matrix Synthesis from Intervertebral Disc Cells
Article Snippet: NP and EP tissues were digested in high-glucose DMEM supplemented with 1M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Invitrogen Co.) supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals), 1M 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES, Sigma-Aldrich), 1M 2-[Bis(2-hydroxyethyl)amino]ethanesulphonic acid (BES, Sigma-Aldrich), 1M HEPES, 1% nonessential amino acids, 2% minimum essential amino acids, sodium bicarbonate, 1% antibiotic-antimyotic, and collagenase (Worthington) for 11 hours at 37°C with stirring.
Enzyme-linked Immunospot:Article Title: Isolation of carbohydrate-specific CD4+ T cell clones from mice after stimulation by two model glycoconjugate vaccines
Article Snippet: .. Penicillin/streptomycin (Cellgro, cat. no. 30002201) Gentamicin (Gibco, cat. no. 15750-060) Sodium bicarbonate (Worthington, cat. no. P8N10891) ACK lysing buffer (Invitrogen, cat. no. A1049201) T cell culture supplement without ConA (TCCS, IL-2 culture supplement; BD Biosciences, cat. no. 354116) Multiscreen HA 96-well ELISpot plate (Millipore, cat. no. MAHAS4510) Anti-mouse IL-2 capture antibody, clone JES6-1A12 (Biolegend, cat. no. 503704) Biotin-labeled anti-mouse IL-2 antibody, clone JES6-5H4 (Biolegend, cat. no. 503804) Anti-mouse IL-4 capture antibody, clone 11B11 (Biolegend, cat. no. 504115) Biotin-labeled anti-mouse IL-4 antibody BVD6-24G2 (Biolegend, cat. no. 504202) Avidin-HRP (Biolegend, cat. no. 405103) AEC ELISpot substrate kit (BD Bioscience, cat. no. 551951) TruStain Fcx anti-mouse CD16/32 antibody (Biolegend, cat. no. 101320) FITC-labeled anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100306) APC anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100312) FITC anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100406) PerCp anti-mouse CD8 antibody, clone 53.67 (Biolegend, cat. no. 100732) PE anti-mouse TCRβ chain antibody, clone H57-597 (Biolegend, cat. no. 109208) Allophycocyanin-labeled anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100412) LEAF purified anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100435) LEAF purified anti-mouse CD8a antibody, clone 53-6.7 (Biolegend, cat. no. 100735) Ethanol Trypan blue stain DMSO Tween-20 .. Vial, 1 dram pH meter Magnetic stir bars Magnetic stir plate PD-10 columns (GE Healthcare, cat. no. 17-0851-01) Sterile syringe-driven filter unit (0.22 µm) Freeze dryer (Freezemobile 12SL) Ultraviolet spectrophotometer (Beckman, cat. no. DU 640) BioLogic HR FPLC system (Bio-Rad) Superdex 200 HiLoad 16/60 prep grade column (GE Healthcare) Dialysis membrane (molecular weight cutoff (MWCO): 12,000–14,000 Da; Spectrum Labs) 96-Well plate, flat-bottomed PowerWave HT microplate reader (Bio-Tek) Criterion TGX any kD stain-free gel (Bio-Rad) Mini-PROTEAN Tetra Cell (Bio-Rad) ChemiDoc MP imager (Bio-Rad)
Modification:Article Title: Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production
Article Snippet: .. Whole lungs and the distal 15 cm of the small intestine were removed from selected mice and incubated in digest media for 45 min followed by the addition of EDTA (2 mM final concentration) for 15 min. Digest medium consisted of Dulbecco’s modified Eagle’s media supplemented with 5% fetal calf serum, sodium bicarbonate, penicillin/streptokinase, 10 mM Hepes, 250 U/mL collagenase I (Worthington Biochemical), 50 U/mL DNase I (Worthington Biochemical), and 0.01% hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) with or without 0.5 mg Brefeldin A (Sigma-Aldrich) in DMSO. .. The samples were filtered and the single cell suspension was treated with Red Blood Cell Lysing Buffer (Sigma-Aldrich) with or without Brefeldin A. APC-, FITC-, or PE-labeled antibodies against murine Mac-3/CD107b (clone M3/84), NK1.1 (clone PK 136), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3ε (clone 145-2C11), CD11b (clone M1/70), CD11c (clone N418), FcεRI (clone MAR-1), anti-mouse Lineage Cocktail (catalog #133305), and isotype controls IgGs (rat and Armenian hamster) were obtained from BioLegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), and/or BD Pharmingen (San Jose, CA, USA).
Staining:Article Title: Isolation of carbohydrate-specific CD4+ T cell clones from mice after stimulation by two model glycoconjugate vaccines
Article Snippet: .. Penicillin/streptomycin (Cellgro, cat. no. 30002201) Gentamicin (Gibco, cat. no. 15750-060) Sodium bicarbonate (Worthington, cat. no. P8N10891) ACK lysing buffer (Invitrogen, cat. no. A1049201) T cell culture supplement without ConA (TCCS, IL-2 culture supplement; BD Biosciences, cat. no. 354116) Multiscreen HA 96-well ELISpot plate (Millipore, cat. no. MAHAS4510) Anti-mouse IL-2 capture antibody, clone JES6-1A12 (Biolegend, cat. no. 503704) Biotin-labeled anti-mouse IL-2 antibody, clone JES6-5H4 (Biolegend, cat. no. 503804) Anti-mouse IL-4 capture antibody, clone 11B11 (Biolegend, cat. no. 504115) Biotin-labeled anti-mouse IL-4 antibody BVD6-24G2 (Biolegend, cat. no. 504202) Avidin-HRP (Biolegend, cat. no. 405103) AEC ELISpot substrate kit (BD Bioscience, cat. no. 551951) TruStain Fcx anti-mouse CD16/32 antibody (Biolegend, cat. no. 101320) FITC-labeled anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100306) APC anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100312) FITC anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100406) PerCp anti-mouse CD8 antibody, clone 53.67 (Biolegend, cat. no. 100732) PE anti-mouse TCRβ chain antibody, clone H57-597 (Biolegend, cat. no. 109208) Allophycocyanin-labeled anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100412) LEAF purified anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100435) LEAF purified anti-mouse CD8a antibody, clone 53-6.7 (Biolegend, cat. no. 100735) Ethanol Trypan blue stain DMSO Tween-20 .. Vial, 1 dram pH meter Magnetic stir bars Magnetic stir plate PD-10 columns (GE Healthcare, cat. no. 17-0851-01) Sterile syringe-driven filter unit (0.22 µm) Freeze dryer (Freezemobile 12SL) Ultraviolet spectrophotometer (Beckman, cat. no. DU 640) BioLogic HR FPLC system (Bio-Rad) Superdex 200 HiLoad 16/60 prep grade column (GE Healthcare) Dialysis membrane (molecular weight cutoff (MWCO): 12,000–14,000 Da; Spectrum Labs) 96-Well plate, flat-bottomed PowerWave HT microplate reader (Bio-Tek) Criterion TGX any kD stain-free gel (Bio-Rad) Mini-PROTEAN Tetra Cell (Bio-Rad) ChemiDoc MP imager (Bio-Rad)
Purification:Article Title: Isolation of carbohydrate-specific CD4+ T cell clones from mice after stimulation by two model glycoconjugate vaccines
Article Snippet: .. Penicillin/streptomycin (Cellgro, cat. no. 30002201) Gentamicin (Gibco, cat. no. 15750-060) Sodium bicarbonate (Worthington, cat. no. P8N10891) ACK lysing buffer (Invitrogen, cat. no. A1049201) T cell culture supplement without ConA (TCCS, IL-2 culture supplement; BD Biosciences, cat. no. 354116) Multiscreen HA 96-well ELISpot plate (Millipore, cat. no. MAHAS4510) Anti-mouse IL-2 capture antibody, clone JES6-1A12 (Biolegend, cat. no. 503704) Biotin-labeled anti-mouse IL-2 antibody, clone JES6-5H4 (Biolegend, cat. no. 503804) Anti-mouse IL-4 capture antibody, clone 11B11 (Biolegend, cat. no. 504115) Biotin-labeled anti-mouse IL-4 antibody BVD6-24G2 (Biolegend, cat. no. 504202) Avidin-HRP (Biolegend, cat. no. 405103) AEC ELISpot substrate kit (BD Bioscience, cat. no. 551951) TruStain Fcx anti-mouse CD16/32 antibody (Biolegend, cat. no. 101320) FITC-labeled anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100306) APC anti-mouse CD3 antibody, clone 145-2C11 (Biolegend, cat. no. 100312) FITC anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100406) PerCp anti-mouse CD8 antibody, clone 53.67 (Biolegend, cat. no. 100732) PE anti-mouse TCRβ chain antibody, clone H57-597 (Biolegend, cat. no. 109208) Allophycocyanin-labeled anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100412) LEAF purified anti-mouse CD4 antibody, clone GK1.5 (Biolegend, cat. no. 100435) LEAF purified anti-mouse CD8a antibody, clone 53-6.7 (Biolegend, cat. no. 100735) Ethanol Trypan blue stain DMSO Tween-20 .. Vial, 1 dram pH meter Magnetic stir bars Magnetic stir plate PD-10 columns (GE Healthcare, cat. no. 17-0851-01) Sterile syringe-driven filter unit (0.22 µm) Freeze dryer (Freezemobile 12SL) Ultraviolet spectrophotometer (Beckman, cat. no. DU 640) BioLogic HR FPLC system (Bio-Rad) Superdex 200 HiLoad 16/60 prep grade column (GE Healthcare) Dialysis membrane (molecular weight cutoff (MWCO): 12,000–14,000 Da; Spectrum Labs) 96-Well plate, flat-bottomed PowerWave HT microplate reader (Bio-Tek) Criterion TGX any kD stain-free gel (Bio-Rad) Mini-PROTEAN Tetra Cell (Bio-Rad) ChemiDoc MP imager (Bio-Rad)
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