5000 HeLa cells/well were seeded in a 96-well plate. Next day cells were transfected in the presence of Lipofectamine2000 with 25 ng of the Renilla reporter, 50 ng of the indicated reporter plasmid, 1000 ng of either pAMC-PROX1 (WT or MUT) or 1000 ng of the corresponding control vector. 36 hours after transfection cells were lysed in 30 µl/well of 1X reporter lysis buffer (Promega). Luciferase activity was measured using a luciferase buffer according to the manufacturer’s instructions (Promega). The assay was repeated two or three times using two biological replicates and the error bars represent SD ± 95%CI across all experiments. For each sample, the firefly luciferase values were normalized to the Renilla luciferase values.
Luciferase buffer
Manufactured by Promega
Sourced in United States
Luciferase buffer is a reagent used to measure luciferase activity in bioluminescence assays. It provides the necessary components for the luciferase-catalyzed oxidation of luciferin, which results in the emission of light. The buffer is designed to optimize the luciferase reaction and ensure reliable and reproducible luminescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1, by Agilent Technologies (9 mentions)
Reporter lysis buffer, by Promega (2 mentions)
Mn protein quantification assay, by Macherey-Nagel (2 mentions)
Luciferase assay tropix lysis solution, by Thermo Fisher Scientific (2 mentions)
Microfuge 22r centrifuge, by Beckman Coulter (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Polyethylenimine reagent, by Polysciences (1 mentions)
Lb 9501 luminometer, by Berthold Technologies (1 mentions)
Clariostar plus microplate reader, by BMG Labtech (1 mentions)
15 protocols using luciferase buffer
1
Luciferase Assay of PROX1 Variants
2
Luciferase Assay for Protein Quantification
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For luciferase assays, approximately 24 hrs after transfection, cells were lysed in a 100 μl Luciferase Assay Tropix Lysis solution (ThermoFisher Scientific), with 0.5 mM DTT. Cells were scraped, transferred to Eppendorf tubes and then centrifuged for 3 minutes at 12,000 g. Twenty μl of the supernatant was used in a 96-well microtiter plate, and a 50 μl luciferase buffer (Promega, Madison, WI, USA) was added. A Luminometer (Labsystem, Luminoscan RT) used to measure lights units. Each experiment was repeated three times with three independent parallels for each experiment, and luciferase values were corrected for protein content in each sample. The total protein concentration was measured using the MN protein quantification assay (Macherey-Nagel GmbH, Düren, Germany).
3
Intracellular Signaling of Anti-CD137 Antibodies
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat #ATCC CCL-119) lentifected with NFkβ (Qiagen; Cat #CLS-013L-1) or SRF (Qiagen; Cat #CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat #E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat #11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
4
Comparative Analysis of Anti-CD137 Antibodies
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat # ATCC CCL-119) lentifected with NFkβ (Qiagen; Cat # CLS-013L-1) or SRF (Qiagen; Cat # CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat # E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat #11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
5
Comparing Anti-CD137 Antibody Signaling
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat# ATCC CCL-119) lentifected with NFkβ (Qiagen; Cat# CLS-013L-1) or SRF (Qiagen; Cat# CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat# E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat#11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
6
Comparative analysis of anti-CD137 agonist signaling
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat# ATCC CCL-119) lentifected with NFκβ (Qiagen; Cat# CLS-013L-1) or SRF (Qiagen; Cat# CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat# E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat#11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
7
Differential Intracellular Signaling by Anti-CD137 Antibodies
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat# ATCC CCL-119) lentifected with NFkβ (Qiagen; Cat# CLS-013L-1) or SRF (Qiagen; Cat# CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat# E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat#11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
8
Differential Intracellular Signaling of Anti-CD137 Antibodies
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat #ATCC CCL-119) lentifected with NFκβ (Qiagen; Cat #CLS-013L-1) or SRF (Qiagen; Cat #CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat #E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat #11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
9
Intracellular Signaling Comparison of Anti-CD137 Antibodies
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Example 24
To further assess the differences between anti-CD137 agonistic antibodies, intracellular signaling was assessed in vitro. Specifically, CCL-119 T cells (ATCC; Cat #ATCC CCL-119) lentifected with NFκβ (Qiagen; Cat #CLS-013L-1) or SRF (Qiagen; Cat #CLS-010L-1) were stimulated with 250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction with varying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotype control. After stimulation for 16 hours in RPMI media without additives, cells were lysed in luciferase buffer (Promega; Cat #E263B) and relative light units (RLUs) were acquired on a BioTek Synergy H1 microplate reader (Cat #11-120-533). Raw RLU data was then exported to Microsoft Excel and fold-induction was calculated by dividing RLUs from stimulated conditions over unstimulated controls.
10
Luciferase Assay: Measuring Transfection Efficiency
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Cells were lysed 24 h post-transfection in 100 µL Luciferase Assay Tropix Lysis solution (Applied Biosystems, Foster City, CA, USA) with DTT added to a final concentration of 0.5 mM (Sigma, St. Louis, MO, USA) freshly added. Cells were scraped and transferred to Eppendorf tubes, followed by 3 min centrifugation at 12,000× g at room temperature in a Microfuge 22R centrifuge (Beckman Coulter, Bea, CA, USA). A 20 µL aliquot of each supernatant was subsequently transferred to 96-well microtiter plate and 50 µL luciferase buffer (Promega, Madison, WI, USA) was added. Light units were measured using CLARIOstar monochromator (520–620 nm) microplate reader (BMG Labtech GmbH, Ortenberg, Germany).
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