Binding Assay:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Sterility:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Luciferase:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Expressing:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Plasmid Preparation:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Microscopy:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Stable Transfection:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Transfection:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
Gene Expression:Article Title: LTBP complex-specific inhibitors of TGFβ and uses thereof
Article Snippet: .. Materials: MvLu1-CAGA12 cells (Clone 4A4) LN229 cell line (high levels of endogenous αVβ8 integrin) Costar white walled TC treated 96 well assay plate #3903 Greiner Bio-One High Binding white μclear 96 well assay plate #655094 Human Fibronectin (Corning #354008) P200 multichannel pipet P20, P200, and P1000 pipets with sterile filter tips for each Sterile microfuge tubes and rack Sterile reagent reservoirs 0.4% trypan blue 2 mL, 5 mL, 10 mL, and 25 mL sterile pipets Tissue culture treated 100 mm or 150 mm plates 70% Ethanol Opti-MEM reduced serum media (Life Tech #31985-070) Lipofectamine 3000 (Life Tech #L3000015) Bright-Glo luciferase assay reagent (Promega #E2620) 0.25% Trypsin+0.53 mM EDTA proTGFb1 expression plasmid, human (SR005) LTBP1S expression plasmid, human (SR044) LTBP3 expression plasmid, human (SR117) LRRC32 (GARP) expression plasmid, human (SR116) LRRC33 expression plasmid, human (SR386) Equipment: BioTek Synergy H1 plate reader TC hood Bench top centrifuge CO2 incubator 37° C. 5% CO2 37° C. water/bead bath Platform shaker Microscope Hemocytometer/countess Definitions: CAGA12 4A4 cells: Derivative of MvLu1 cells (Mink Lung Epithelial Cells), stably transfected with CAGA12 synthetic promoter, driving luciferase gene expression DMEM-0.1% BSA: Assay media; base media is DMEM (Gibco Cat #11995-065), media also contains BSA diluted to 0.1% w/v, penicillin/streptinomycin, and 4 mM glutamine D10: DMEM 10% FBS, P/S, 4 mM glutamine, 10% NEAA, 1× GlutaMAX (Gibco Cat #35050061) CAGA12 (4A4) media: D10+0.75 ug/mL puromycin Procedure: On Day 0, integrin expressing cells were seeded for transfection. ..
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