Flow Cytometry:Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor
Article Snippet: .. Abs used For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD8-PE (A07757; Beckman Coulter, Brea, CA), anti–CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti–CD8-PE (A07757; Beckman Coulter), anti–CD57-FITC (322306; BioLegend, San Diego, CA), anti–CD85j-allophycocyanin (333720; BioLegend), CTLA-4–allophycocyanin (349908; BioLegend), anti–PD-1-PE ( B30634 ; Beckman Coulter), anti-FMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti–MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. Reagents The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-γ ELISA kit ( Ab100537 ; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect d -luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD8-PE (A07757; Beckman Coulter, Brea, CA), anti–CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti–CD8-PE (A07757; Beckman Coulter), anti–CD57-FITC (322306; BioLegend, San Diego, CA), anti–CD85j-allophycocyanin (333720; BioLegend), CTLA-4–allophycocyanin (349908; BioLegend), anti–PD-1-PE ( B30634 ; Beckman Coulter), anti-FMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti–MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-γ ELISA kit ( Ab100537 ; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect d -luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Article Title: Blockade of N -Glycosylation Promotes Antitumor Immune Response of T Cells.
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD3-FITC (A07746; Beckman Coulter), anti–CD8-PE (A07757; Beckman Coulter), anti-CD56- allophycocyanin (362504; BioLegend), anti–CD244(2B4)-PE/Cy7 (329519; BioLegend), anti– CD94-FITC (305504; BioLegend), anti–thymocyte selection-associated high-mobility group box protein (TOX) (682601; BioLegend), antiEomesoderm (EOMES) (662001; BioLegend), anti–T-bet (644801; BioLegend), anti–CD107a(LAMP1)-allophycocyanin (328619; BioLegend), anti–CD8-PC5 (A07758; Beckman Coulter), anti–CD314(NKG2D)-PE (A08934; Beckman Coulter), anti–MHC class I chain-related protein (MIC) A/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems), anti-ULBP2/5/6 (MAB1298; R&D Systems), anti-ULBP3 (MAB1517; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), goat anti-mouse IgG-FITC (115-096-146; Jackson Immunoresearch), anti–CD197(CCR7)-FITC (353215; BioLegend), anti-CD45ROPE/Cy5 (304208; BioLegend), anti-Galectin3 (LS-C140105; LifeSpan BioSciences), anti–TCRab-PC7 (306720; BioLegend), anti–TCRgd-FITC (331208; BioLegend), anti–CD45RA-FITC (A07786; Beckman Coulter), and anti-CD62L (IM2114; Beckman Coulter). .. The following reagents were used: a-galactosylceramide (aGalCer) (67576; Sigma-Aldrich) 2DG (D8735; Sigma-Aldrich), Human Cytokine G1 27- plex Panel (M500KCAFOY; Bio-Rad laboratories), Human Perforin ELISA Kit (ab46068; Abcam), Human Granzyme B ELISA Kit (ab46142; Abcam), CellstainR–calcein-AM solution (341-07901; Dojindo), CytoRed solution (C410-10; Dojindo), sodium oxamate (O2751; Sigma-Aldrich), bromopyruvic acid (Sigma-Aldrich), D-mannose (M2069; Sigma-Aldrich), L-mannose (M1308; Tokyo Kasei Kogyou), IntraPrep Permeabilization Reagent (A07803; Beckman Coulter), IL-2 (87890; Nipro), Human IFN-g ELISA Kit (Ab100537; Abcam), Recombinant Galectin 3 (P734-1; BBI Solutions), lactose (L3750; Sigma-Aldrich), Annexin V–FITC Apoptosis Detection Kit (15342-54; Nacalai Tesque), EasySep Human CD8 Positive Selection Kit II (17953; STEMCELL Technologies), and EasySep Human CD4 Positive Selection Kit II (17952; STEMCELL Technologies).
Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N-Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor.
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti CD8-PE (A07757; Beckman Coulter, Brea, CA), anti CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti CD8-PE (A07757; Beckman Coulter), anti CD57-FITC (322306; BioLegend, San Diego, CA), anti CD85j-allophycocyanin (333720; BioLegend), CTLA-4 allophycocyanin (349908; BioLegend), anti PD-1-PE (B30634; Beckman Coulter), antiFMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-g ELISA kit (Ab100537; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect D-luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Western Blot:Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor
Article Snippet: .. Abs used For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD8-PE (A07757; Beckman Coulter, Brea, CA), anti–CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti–CD8-PE (A07757; Beckman Coulter), anti–CD57-FITC (322306; BioLegend, San Diego, CA), anti–CD85j-allophycocyanin (333720; BioLegend), CTLA-4–allophycocyanin (349908; BioLegend), anti–PD-1-PE ( B30634 ; Beckman Coulter), anti-FMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti–MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. Reagents The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-γ ELISA kit ( Ab100537 ; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect d -luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N -Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD8-PE (A07757; Beckman Coulter, Brea, CA), anti–CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti–CD8-PE (A07757; Beckman Coulter), anti–CD57-FITC (322306; BioLegend, San Diego, CA), anti–CD85j-allophycocyanin (333720; BioLegend), CTLA-4–allophycocyanin (349908; BioLegend), anti–PD-1-PE ( B30634 ; Beckman Coulter), anti-FMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti–MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-γ ELISA kit ( Ab100537 ; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect d -luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Article Title: Blockade of N -Glycosylation Promotes Antitumor Immune Response of T Cells.
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD3-FITC (A07746; Beckman Coulter), anti–CD8-PE (A07757; Beckman Coulter), anti-CD56- allophycocyanin (362504; BioLegend), anti–CD244(2B4)-PE/Cy7 (329519; BioLegend), anti– CD94-FITC (305504; BioLegend), anti–thymocyte selection-associated high-mobility group box protein (TOX) (682601; BioLegend), antiEomesoderm (EOMES) (662001; BioLegend), anti–T-bet (644801; BioLegend), anti–CD107a(LAMP1)-allophycocyanin (328619; BioLegend), anti–CD8-PC5 (A07758; Beckman Coulter), anti–CD314(NKG2D)-PE (A08934; Beckman Coulter), anti–MHC class I chain-related protein (MIC) A/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems), anti-ULBP2/5/6 (MAB1298; R&D Systems), anti-ULBP3 (MAB1517; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), goat anti-mouse IgG-FITC (115-096-146; Jackson Immunoresearch), anti–CD197(CCR7)-FITC (353215; BioLegend), anti-CD45ROPE/Cy5 (304208; BioLegend), anti-Galectin3 (LS-C140105; LifeSpan BioSciences), anti–TCRab-PC7 (306720; BioLegend), anti–TCRgd-FITC (331208; BioLegend), anti–CD45RA-FITC (A07786; Beckman Coulter), and anti-CD62L (IM2114; Beckman Coulter). .. The following reagents were used: a-galactosylceramide (aGalCer) (67576; Sigma-Aldrich) 2DG (D8735; Sigma-Aldrich), Human Cytokine G1 27- plex Panel (M500KCAFOY; Bio-Rad laboratories), Human Perforin ELISA Kit (ab46068; Abcam), Human Granzyme B ELISA Kit (ab46142; Abcam), CellstainR–calcein-AM solution (341-07901; Dojindo), CytoRed solution (C410-10; Dojindo), sodium oxamate (O2751; Sigma-Aldrich), bromopyruvic acid (Sigma-Aldrich), D-mannose (M2069; Sigma-Aldrich), L-mannose (M1308; Tokyo Kasei Kogyou), IntraPrep Permeabilization Reagent (A07803; Beckman Coulter), IL-2 (87890; Nipro), Human IFN-g ELISA Kit (Ab100537; Abcam), Recombinant Galectin 3 (P734-1; BBI Solutions), lactose (L3750; Sigma-Aldrich), Annexin V–FITC Apoptosis Detection Kit (15342-54; Nacalai Tesque), EasySep Human CD8 Positive Selection Kit II (17953; STEMCELL Technologies), and EasySep Human CD4 Positive Selection Kit II (17952; STEMCELL Technologies).
Article Title: Efficacy against Lung Cancer Is Augmented by Combining Aberrantly N-Glycosylated T Cells with a Chimeric Antigen Receptor Targeting Fragile X Mental Retardation 1 Neighbor.
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti CD8-PE (A07757; Beckman Coulter, Brea, CA), anti CD4-FITC (53-0048-42; eBioscience, San Diego, CA), anti CD8-PE (A07757; Beckman Coulter), anti CD57-FITC (322306; BioLegend, San Diego, CA), anti CD85j-allophycocyanin (333720; BioLegend), CTLA-4 allophycocyanin (349908; BioLegend), anti PD-1-PE (B30634; Beckman Coulter), antiFMR1NB (H00158521-W01P; Abnova, Taipei, Taiwan), anti MAGE-A (6C1) Alexa Fluor 546 (sc-20034 AF546; Santa Cruz Biotechnology, Dallas, TX), anti-MICA/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems, Minneapolis, MN), anti-ULBP2/5/6 (MAB1298; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), and goat anti-mouse IgG-FITC (115-096-146; Jackson ImmunoResearch Laboratories, West Grove, PA). .. The following reagents were used: ALyS505N-175 (Cell Science & Technology Institute, Sendai, Japan), annexin V-FITC apoptosis detection kit (15342-54; Nacalai Tesque, Kyoto, Japan), Applied Biosystems Power SYBR Green PCR master mix (4309155; Thermo Fisher Scientific, Waltham, MA), biotin labeling kit-NH2 (LK03; Dojindo Molecular Technologies, Rockville, MD), endogenous biotin blocking kit (760-050; Ventana Medical Systems, Tucson, AZ), BLOCK-iT polymerase II miR-RNA interference expression vector kits (Invitrogen, Carlsbad, CA), Dako REAL endogenous peroxidase blocking solution (S202386-2; Agilent Technologies, Santa Clara, CA), Exi293 expression system (A14635; Thermo Fisher Scientific), HisLink protein purification systems (V1320; Promega, Wisconsin, WI), human lung cancer and normal tissue array (LC812; US Biomax, Derwood, MD), human T cell Nucleofector kit (VPA-1002; Lonza, Basel, Switzerland), Lenti-X concentrator (631231; Clontech Laboratories, Mountain View, CA), One-Step luciferase assay system (60690-1; New England Biolabs, Ipswich, MA), recombinant human PD-L1/B7-H1 Fc chimera biotin protein (CF BT156-050; R&D Systems), recombinant human PD-L1 protein (Active) (ab167713; Abcam, Cambridge, U.K.), recombinant human CD86 protein (Fc chimera) (ab167720; Abcam), SuperScript III first-strand synthesis system for RT-PCR (18080-051; Invitrogen), Ph.D.-C7C phage display peptide library kit (E8120S; New England Biolabs), human IFN-g ELISA kit (Ab100537; Abcam), human IL-2 ELISA kit (ab270883; Abcam), RetroNectin (T100B; Takara Bio, Kusatsu, Japan), streptavidin-HRP (ab7403; Abcam), streptavidin-FITC (405201; BioLegend), XenoLight RediJect D-luciferin ultra (770505; PerkinElmer, Richardson, TX), TRIzol reagent (15596-026; Invitrogen), ViraPower lentiviral expression systems (K367-20; Invitrogen), tumor dissociation kit (130-095-929; Miltenyi Biotec, Mountain View, CA), and XTT cell proliferation assay kit (10010200; Cayman Chemical, Ann Arbor, MI).
Selection:Article Title: Blockade of N -Glycosylation Promotes Antitumor Immune Response of T Cells.
Article Snippet: .. For flow cytometry and immunoblotting, the following primary Abs were used: anti–CD3-FITC (A07746; Beckman Coulter), anti–CD8-PE (A07757; Beckman Coulter), anti-CD56- allophycocyanin (362504; BioLegend), anti–CD244(2B4)-PE/Cy7 (329519; BioLegend), anti– CD94-FITC (305504; BioLegend), anti–thymocyte selection-associated high-mobility group box protein (TOX) (682601; BioLegend), antiEomesoderm (EOMES) (662001; BioLegend), anti–T-bet (644801; BioLegend), anti–CD107a(LAMP1)-allophycocyanin (328619; BioLegend), anti–CD8-PC5 (A07758; Beckman Coulter), anti–CD314(NKG2D)-PE (A08934; Beckman Coulter), anti–MHC class I chain-related protein (MIC) A/B (320910; BioLegend), anti-ULBP1 (MAB1380; R&D Systems), anti-ULBP2/5/6 (MAB1298; R&D Systems), anti-ULBP3 (MAB1517; R&D Systems), mouse IgG1 (400124; BioLegend), mouse IgG2a (400202; BioLegend), goat anti-mouse IgG-FITC (115-096-146; Jackson Immunoresearch), anti–CD197(CCR7)-FITC (353215; BioLegend), anti-CD45ROPE/Cy5 (304208; BioLegend), anti-Galectin3 (LS-C140105; LifeSpan BioSciences), anti–TCRab-PC7 (306720; BioLegend), anti–TCRgd-FITC (331208; BioLegend), anti–CD45RA-FITC (A07786; Beckman Coulter), and anti-CD62L (IM2114; Beckman Coulter). .. The following reagents were used: a-galactosylceramide (aGalCer) (67576; Sigma-Aldrich) 2DG (D8735; Sigma-Aldrich), Human Cytokine G1 27- plex Panel (M500KCAFOY; Bio-Rad laboratories), Human Perforin ELISA Kit (ab46068; Abcam), Human Granzyme B ELISA Kit (ab46142; Abcam), CellstainR–calcein-AM solution (341-07901; Dojindo), CytoRed solution (C410-10; Dojindo), sodium oxamate (O2751; Sigma-Aldrich), bromopyruvic acid (Sigma-Aldrich), D-mannose (M2069; Sigma-Aldrich), L-mannose (M1308; Tokyo Kasei Kogyou), IntraPrep Permeabilization Reagent (A07803; Beckman Coulter), IL-2 (87890; Nipro), Human IFN-g ELISA Kit (Ab100537; Abcam), Recombinant Galectin 3 (P734-1; BBI Solutions), lactose (L3750; Sigma-Aldrich), Annexin V–FITC Apoptosis Detection Kit (15342-54; Nacalai Tesque), EasySep Human CD8 Positive Selection Kit II (17953; STEMCELL Technologies), and EasySep Human CD4 Positive Selection Kit II (17952; STEMCELL Technologies).
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