LTR basal activity from strains A4 [21 (link)], KV1772 [43 (link)], Ov496 [19 (link)] and CAEV-Co [42 (link)] was assayed using a luciferase reporter system. Briefly, DSF, FSF and GSM cells (105 in 24-well plates) were transfected with 200 ng of each LTR construction using 4 µL of Lipofectamine-LTX Reagent and 0.2 µL of PLUS-Reagent (Life Technologies). Plasmids pGL4.13 [luc2/SV40] and “empty” p-GL4.10 [luc2] were used as positive and negative controls, respectively. At the same time, 20 ng of pGL4.73 [hRluc/SV40] Vector (Promega) were cotransfected, so that the firefly activity was standardized according to the Renilla luciferase activity. After 24 h, cells were harvested with Passive Lysis 5X Buffer (Promega), and firefly and renilla luciferase activity were measured following manufacturer’s instructions. Results were expressed as: Relative Luminiscence Units (RLU) firefly luminescence/RLU Renilla luminescence.
Passive lysis 5x buffer
Manufactured by Promega
Sourced in United States
Passive Lysis 5X Buffer is a concentrated buffer solution designed for cell lysis. When diluted, it facilitates the release of cellular contents for subsequent analysis or extraction.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual glo luciferase assay kit, by Promega (2 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Pgl4.73 hrluc sv40 vector, by Promega (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
Jetpei transfection reagent, by Polyplus Transfection (1 mentions)
Phrl tk, by Promega (1 mentions)
Dual luciferase assay kit, by Vazyme (1 mentions)
Anti pcna, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Affinity Biosciences (1 mentions)
Tanon 5200 chemiluminescent imaging system, by Shanghai Tanon Science & Technology (1 mentions)
Anti gapdh, by Affinity Biosciences (1 mentions)
Super ecl detection reagent, by Yeasen (1 mentions)
Blocking one p, by Nacalai Tesque (1 mentions)
Multi gauge 3, by Fujifilm (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
10 protocols using passive lysis 5x buffer
1
Comparative Analysis of LTR Basal Activity
2
Dual-Luciferase Assay of TLR5 Activation
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For the dual-luciferase assays, HEK293 cells were seeded in 96-well plates (Corning) at 2–3 × 104 cells per well (0.1 ml). The next day, the cells were transiently transfected with plasmids expressing wtTLR5 or chimeric constructs, pELAM-1 (C. Kirschning, University of Duisburg-Essen, Germany) expressing NF-κB-dependent firefly luciferase (50 ng per well), and phRL-TK (Promega) constitutively expressing Renilla luciferase (5 ng per well) using the jetPEI transfection reagent (Polyplus Transfection). The total amount of DNA for each transfection was kept constant by adding appropriate amounts of control plasmid pcDNA3 (Invitrogen). After 24 h, the cells were either lysed or the medium was changed and the cells were stimulated with purified recombinant flagellin (10 μl) for an additional 18 h before lysis. The cells were lysed in Passive Lysis 5x Buffer (Promega) and analyzed for reporter gene activities using a dual-luciferase reporter assay. Each experiment was repeated at least three times, and each measurement was performed in at least four biological parallels. A student’s unpaired two-tailed t-test was used for statistical comparison.
3
Quantifying SCD1 Promoter Activity
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To assess SCD1 gene promotor activity, cells were co-transfected with SCD1 luciferase reporter plasmid, with Renilla control reporter as an internal control. After 48 h, cells were lysed in a Passive Lysis 5X Buffer (Promega, E1941), and the enzymatic activity of luciferase were measured using a Dual-Luciferase Assay kit (Vazyme, DL101-01), according to the manufacturer’s protocol. To construct SCD1 luciferase reporter plasmid, the 1547-bp sequence above the TSS site of SCD1 was inserted in a pGL3-Basic vector using the specific enzymes at KpnI and XhoI sites.
4
Western Blot Analysis of Protein Expression
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The MDS cells were transfected with the indicated oligonucleotides and plasmids for 48 h. Then, cells were lysed with Passive Lysis 5X Buffer (Promega Corporation, WI 53,711 USA). On a 10% SDS-PAGE gel, cell extracts were processed and blotted on polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% w/v non-fat milk, the membranes were incubated overnight at 4°C with primary antibodies including anti-PCNA (Affinity Biosciences, USA; dilution 1:5000), anti-HSPA12B (Affinity Biosciences, USA; dilution 1:1000), anti-GAPDH (Affinity Biosciences, USA; dilution 1:1000). Then, the membranes were further probed with HRP-conjugated secondary antibodies (Affinity Biosciences, USA; dilution 1:5000). The protein bands were viewed using Super ECL Detection Reagent (Yeasen Biotech Co., Ltd. Shanghai, China) on a Tanon‑5200 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd. Shanghai, China) and the band intensity of protein was measured by ImageJ software[36 ].
5
Estrogen Receptor Transactivation Assay
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MCF7 cells were seeded in a 24-well plate at 5 x 104 cells per well in phenol-free DMEM with 10% DCFCS and 100 U penicillin/0.1 mg ml-1 streptomycin and 2mM L-glutamine. After 24 hours of incubation, transfection of plasmid DNA was performed using Lipofectamine 3000 (Invitrogen; L3000015). Cells were transfected with 100ng of ERE_Luciferase reporter, 10ng of the renilla luciferase control plasmid (pRL-CMV), 10ng of pSG5_ER-α, 15 nm of siRNA and 280ng of Bluescribe DNA (BSM) per well; totalling 400ng of DNA/well. After 12 hours of transfection the media was replaced with fresh phenol-free DMEM with 10% DCFCS and 100 U penicillin/0.1 mg ml-1 streptomycin and 2mM L-glutamine. Treatment with 10-8 17-β-estradiol (SIGMA E8875) or control treatment was administered and the cells incubated for 24 hours. Cell lysates are then obtained using Passive lysis 5X buffer (Promega; E1941). The firefly and renilla luciferase activity was determined using DualGlo luciferase assay kit (Promega; E2920) according to the manufacturer protocol. The renilla luciferase activity measurement was utilised as control for transfection efficiency and therefore the ERE_Luciferase activity was normalised to the reading obtained for the renilla luciferase activity.
6
Western Blotting for Protein Detection
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Western blotting was performed as described previously [14 (link)]. The cells were washed with PBS, lysed in Passive Lysis 5x buffer (Promega, Madison, WI, USA), and supplemented with a protease inhibitor cocktail (Nacalai Tesque). Protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan). The membranes were incubated with Blocking One-P (Nacalai Tesque) and probed with the following primary antibodies: anti-GAPDH (14C10) (1 : 1000, #2118; Cell Signaling Technology, Danvers, MA), anti-PKR (B-10) (1 : 1000, sc-6282; Santa Cruz Biotechnology, Dallas, TX), and anti-PKR (phospho T446) [E120] (1 : 200, ab32036; Abcam). Blotted membranes were detected using Chemi-Lumi One Super (Nacalai Tesque) and visualized using densitometry on an Amersham ImageQuant 800 platform (Cytiva, Tokyo, Japan). Immunoblots were quantified using Multi Gauge 3.1 software (FUJIFILM, Tokyo, Japan).
7
Estrogen Receptor Transactivation Assay
8
Quantifying Cellular Reactive Oxygen Species
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5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen GmbH) was used as an indicator for reactive ROS in cells. CM-H2DCFDA was reconstituted at 5 mM in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and then diluted with Hank's balanced salt solution (HBSS) (PAA Laboratories GmbH) to 5 μM. Cells were washed twice with HBSS, incubated with 5 μM CM-H2DCFDA for 45 min at 37°C, followed by further washing. Passive Lysis Buffer 5x (Promega) was diluted 1:5 with distilled water and protease inhibitor cocktail (Roche Diagnostics GmbH). CM-H2DCFDA-labeled cells were lysed in 400 μl lysis buffer. After centrifugation, 100 μl of the supernatant were added to 96-well tissue culture plate. Resulting fluorescence was monitored using a microplate reader (Infinite M200, Tecan) with excitation and emission wavelengths of 495 and 520 nm, respectively. Signal was quantified as relative fluorescence units (RFU).
9
Luciferase Reporter Assay for Promoter Activity
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All cell lines were seeded in 24-well plates at a density of 105 cells per well; 24 h later, they were transfected with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) using 1 μg of plasmid DNA and 2 μg of Lipofectamine per well. Five hours later, medium was refreshed and cells were maintained for 48 h before addition of the Passive Lysis Buffer 5X (Promega, Madison, WI). Luciferase activity was measured with the Luciferase Reporter Assay System (Promega) in a Luminat KB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Data were normalized by protein content in each sample (in μg), determined by the Bradford assay (Bio-Rad, Hercules, CA). Promoter activity is represented as percentage of luciferase activity, using the CMV promoter as a reference.
10
OTC Protein Expression in HEK293T Cells
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The expression of OTC protein was performed in HEK293T cells which lack endogenous OTC expression. HEK293T cells were maintained in Dulbecco's modified Eagle's medium high glucose, pyruvate, and supplemented with 10% fetal bovine serumand 5% Penicillin/Streptomycin. Cells were seeded at a density of 3 × 105 cells/well in 24‐well plates. After 24 h, cells were transfected using 1.5 µg selected OTC plasmid (pIRES‐AEGFP‐OTC) and Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific) in Opti‐MEM reduced serum medium (Gibco, Thermo Fisher Scientific) according to the manufacturer's guidelines. 24 h after transfection, cells were inspected in the fluorescent cell imager ZOE™ (Bio‐RAD) to calculate transfection efficiency, by counting the number of cells expressing enhanced green fluorescent protein in relation to the total number of cells in the same field and no significant transfection efficiency differences were observed between backgrounds. Immediately after, cells were collected using passive lysis buffer 5X (Promega) supplemented with 10 µl protease inhibitor, and stored at −80°C. For each genetic background, three independent replicate assays were performed.
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