101 L cells were seeded in triplicate at 2.2 × 104 cells/well on a clear 48-well tissue culture plate in phenol red-free DMEM with 5% charcoal-stripped FBS. After 24 hours, media were removed and replaced with media containing 0.1% dimethyl sulfoxide (DMSO) or a range of AF doses (100nM, 500nM, 1 μM, 10 μM). After 18 hours of compound treatment, the cells were washed with 50 μL 1× PBS (Gibco, Invitrogen) and lysed with 50 μL Tropix lysis buffer (100 mM K2HPO4, 0.2% Triton X-100, pH 7.8, Applied Biosystems). Cell lysate was mixed 1:1 with luciferase substrate (Promega, Madison, WI), and luminescence was measured with a 700-nm filter on a Victor X5 microplate reader (PerkinElmer, Waltham, MA). The Bradford method (Bio-Rad) was used to measure total protein in each sample. Raw luciferase data was normalized to both total protein and background luciferase expression in the DMSO control samples and expressed as fold-increase over DMSO.
Tropix lysis buffer
Manufactured by Thermo Fisher Scientific
Tropix Lysis Buffer is a reagent used for the extraction and purification of cellular components, such as proteins and nucleic acids, from biological samples. It is designed to effectively lyse cells and solubilize their contents, enabling the isolation and analysis of the desired biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
24 well plate, by Corning (2 mentions)
Safire plate reader, by Tecan (2 mentions)
Tropix galactostar reaction mixture, by Thermo Fisher Scientific (2 mentions)
Luciferase substrate, by Promega (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Victor x5 microplate reader, by PerkinElmer (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Pgl3 luciferase reporter plasmid, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
12 well plate, by Corning (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
K3fe cn 6, by Merck Group (1 mentions)
6 protocols using tropix lysis buffer
1
Luciferase Assay for Compound Screening
2
Transient Transfection and Luciferase Assay
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HEK 293T cells were split into 12 wells and transiently transfected with the plasmids to be investigated in triplicates along with the luciferase reporter construct and a β-galactosidase expression vector. After 24hrs of transfection, the cells were lysed with Tropix lysis buffer (Applied Biosystems) for 15min at room temperature. Lysates were centrifuged at 15000g for 5min. Supernatant was transferred to a 96 well plate and luciferase activity was measured and normalised to β-galactosidase activity to correct for transfection efficiency.
3
SOX5 Variant Transcriptional Regulation
4
Cloning and Characterization of CDKN2B Promoter
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The CDKN2B promoter fragment containing seven CpG sites was generated using PCR as aforementioned, and the primer sequences of CDKN2B were as follows: Forward, 5′-GGGGCAGTGAGGACT-3′ and reverse, 5′-GCCTGGATTGCTTCT-3′. The subsequent PCR product was cloned into pCR2.1 (included in the kit) using a T-A Cloning Kit (Invitrogen; Thermo Fisher Scientific, Inc.) and sequenced. Plasmids containing CDKN2B promoter region were amplified and digested with XhoI and KpnI (New England Biolabs, Ipswich, MA, USA). The target DNA fragment containing the reporter gene was cloned into pGL3-Luciferase reporter plasmid (Promega Corporation, Madison, WI, USA). Subsequently, constructed pGL3-CDKN2B-Luciferase plasmid was transfected into 293 cells using Lipofectamine 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. A total of 24 h following transfection, cells were lysed with Tropix lysis buffer (Applied Biosystems; Thermo Fisher Scientific, Inc.). Luciferase and β-galactosidase activities were measured using a Luciferase Assay System (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. β-Galactosidase activity was used to normalize transfection efficiency.
5
Quantifying Viral Transduction Efficiency
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HeLa or D17 cells were seeded in 24-well plates (Corning) at densities of 2.5x104 cells/well and 2x104 cells/well, respectively. Cells were infected 24 h later, with WT and mutant VLPs normalised on their RT activity and incubated at 37°C for 72 h. Cells were then lysed in Tropix Lysis buffer (Thermo Fisher Scientific) and frozen at -20°C. To measure LacZ activity, cell lysate was mixed with Tropix galactostar reaction mixture (Thermo Fisher Scientific) and luminescence was measured for 1 h at 10 min intervals on a Tecan Safire plate reader.
Absolute infectious titres of VLPs for microscopy assays were determined by X-gal staining of infected HeLa cells. HeLa cells were seeded in 12-well plates (Corning) at a density of 5x104 cells/well and infected 24 h later with a 10-fold dilution series of VLPs by spinoculation (1600 g, 2 h, 16°C). Cells were then incubated at 37°C for 30 min prior to replacement of media with warm serum-supplemented DMEM. After 72 h, cells were washed in PBS and fixed for 10 min with 2% formaldehyde and 0.2% glutaraldehyde. Staining was performed overnight at 37°C in PBS with 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (Sigma), 4 mM K3Fe(CN)6 (Sigma), 4mM K4Fe(CN)6ˑ3H2O (Sigma), 2mM MgCl2 (Thermo). The number of blue LacZ-expressing colonies were counted using a light microscope (Olympus) and the viral titre calculated.
Absolute infectious titres of VLPs for microscopy assays were determined by X-gal staining of infected HeLa cells. HeLa cells were seeded in 12-well plates (Corning) at a density of 5x104 cells/well and infected 24 h later with a 10-fold dilution series of VLPs by spinoculation (1600 g, 2 h, 16°C). Cells were then incubated at 37°C for 30 min prior to replacement of media with warm serum-supplemented DMEM. After 72 h, cells were washed in PBS and fixed for 10 min with 2% formaldehyde and 0.2% glutaraldehyde. Staining was performed overnight at 37°C in PBS with 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (Sigma), 4 mM K3Fe(CN)6 (Sigma), 4mM K4Fe(CN)6ˑ3H2O (Sigma), 2mM MgCl2 (Thermo). The number of blue LacZ-expressing colonies were counted using a light microscope (Olympus) and the viral titre calculated.
6
HeLa Cell-based Viral Infection Assay
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