A2 and PSM cells were seeded in 12 well plates at a density of 2×105 and 8×104 cells per well, respectively. On the next day (d0), cells were transfected in conditioned medium using FuGENE® HD Transfection Reagent (Promega). For luciferase assays using BAC derived reporter constructs, firefly and renilla luciferase reporter constructs were cotransfected in a 2:1 molar ratio. For transfection of one well of a 12 well plate, 1 μg of the respective firefly luciferase BAC reporter construct was used. After 16-18 h (d1), medium was changed. On d2, cells were transferred to a 6 cm plate. On d7, cells were harvested in 50 μl of 1x PLB (Promega). For plasmid based luciferase reporter systems, firefly and renilla luciferase constructs were cotransfected in a 5:1 molar ratio using 1 μg total plasmid DNA per well. Medium was changed after 16-18 h, and on d2, cells were harvested in 60 μl of 1× PLB (Promega). Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega) and the TriStar LB941 microplate multimode reader (Berthold Technologies). For each experiment, cells were seeded and transfected in triplicates. Unless noted otherwise, data are presented as mean ± standard deviation of three or four independently performed experiments and significance was determined using Welch’s t-test (*: p < 0.05, **: p < 0.01, ***: p < 0.001, n.s.: not significant).
Passive lysis buffer (plb)
Manufactured by Promega
Sourced in United States, Germany, United Kingdom, France, Japan, China, Switzerland, Italy, Canada
Passive lysis buffer is a solution used for the gentle lysis of cells to extract proteins or other biomolecules. It facilitates the release of cellular contents without denaturing the target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (531 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (306 mentions)
Luciferase assay system, by Promega (130 mentions)
Dual luciferase assay kit, by Promega (100 mentions)
Dual luciferase assay system, by Promega (99 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (95 mentions)
Dual luciferase reporter assay, by Promega (84 mentions)
Dual luciferase reporter assay kit, by Promega (69 mentions)
Prl tk, by Promega (69 mentions)
Glomax 20 20 luminometer, by Promega (68 mentions)
Luciferase assay reagent, by Promega (59 mentions)
Opti mem, by Thermo Fisher Scientific (51 mentions)
Dual glo luciferase assay system, by Promega (41 mentions)
Dual luciferase assay, by Promega (35 mentions)
Glomax luminometer, by Promega (35 mentions)
Glomax 96 microplate luminometer, by Promega (34 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (32 mentions)
Dual luciferase kit, by Promega (30 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (30 mentions)
Dual luciferase reporter system, by Promega (29 mentions)
2 573 protocols using passive lysis buffer (plb)
1
Luciferase Reporter Assay Protocol
2
Optimized Extraction for Dual Reporter Assays
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Several protocols were tested for the extraction of leaf tissue to identify a procedure that is compatible with both glucuronidase and luciferase activity measurements, so that all reporter enzymes could be analyzed for the same extract. The harvested, frozen leaf discs were ground to powder with liquid N2 and then 9 μl extraction buffer was added per mg tissue (300 μl buffer for six ~6 mm leaf discs). As extraction buffer we tried either GUS extraction buffer (25 mM potassium phosphate pH7.8, 1 mM EDTA, 7 mM 2-mercaptoethanol, 1% Triton X-100, and 10% glycerol) or 1X Cell Culture Lysis Reagent (CCLR; Luciferase assay systems, Promega). The glucuronidase and luciferase activities determined for the extracts were more consistent and higher when CCLR had been used for the preparation and PLB (Promega) for the dilution of the extracts, and therefore CCLR and PLB were used for all further experiments. Extracts were incubated on ice for 1 hour and cell debris was pelleted by centrifugation for 10 min at 13000 × g (SpeedFuge ® SFR13K, Savant). The supernatant was then diluted 75x with PLB and used for protein, glucuronidase and luciferase assays.
3
In Vitro Translation of Luciferase Reporters
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In vitro translation was typically performed in a 10 μl reaction mixture containing 5 μl of RRL supernatant, 22.6 nM of purified ribosome, 1.1 nM of reporter mRNA, 75 mM KCl, 0.75 mM MgCl2, and 20 µM of amino acid mixture (Promega).
For the Fluc reporter, the reaction mixture was incubated at room temperature for 90 min and stopped by the addition of 20 μl of 1× Passive Lysis Buffer (Promega). The FLuc luminescence from 30 μl of the mixture was measured with the Dual-Luciferase Reporter Assay System (Promega).
For Rluc-Y0×-Fluc and Rluc-Y39×-Fluc reporters, the reaction mixture above was scaled up to 120 μl and incubated at room temperature. 10 μl of the reaction mixture was taken as an aliquot every 5 min and mixed with 20 μl of 1× Passive Lysis Buffer (Promega) to stop the reaction. The Rluc and FLuc luminescence from 30 μl of the mixture was measured with the Dual-Luciferase Reporter Assay System (Promega). The luminescences at 20–30 min were used to calculate the slope of Rluc and Fluc synthesis.
For the Fluc reporter, the reaction mixture was incubated at room temperature for 90 min and stopped by the addition of 20 μl of 1× Passive Lysis Buffer (Promega). The FLuc luminescence from 30 μl of the mixture was measured with the Dual-Luciferase Reporter Assay System (Promega).
For Rluc-Y0×-Fluc and Rluc-Y39×-Fluc reporters, the reaction mixture above was scaled up to 120 μl and incubated at room temperature. 10 μl of the reaction mixture was taken as an aliquot every 5 min and mixed with 20 μl of 1× Passive Lysis Buffer (Promega) to stop the reaction. The Rluc and FLuc luminescence from 30 μl of the mixture was measured with the Dual-Luciferase Reporter Assay System (Promega). The luminescences at 20–30 min were used to calculate the slope of Rluc and Fluc synthesis.
4
Validating miRNA-Gene Interactions
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A dual-luciferase report assay was used for the validation of the miRNA-gene pairs. The luciferase reporter plasmid was built using the pmirGLO vector, into which the wild-type and mutant candidate genes were cloned. For this purpose, an appropriate number of 293T cells were seeded into a 12-well plate and cultured at 37°C in an incubator overnight. Cells were co-transfected with the luciferase reporter plasmids and miRNA mimics/mimics control using Lipofectamine 2000 (Invitrogen), and the efficiency of transfection was assessed. After 24 h, Firefly and Renilla luciferase activity were detected with a Dual-Luciferase Reporter Assay kit (PR-E1910, Promega, Wisconsin, USA). Relative luciferase activity was defined as the ratio of Renilla luciferase activity to the Firefly luciferase activity with that of the control set as 1.0. Briefly, cells were washed with phosphate-buffered saline, and 400 μL of 1×Passive Lysis Buffer (Promega) was added to the cultured well and shaken gently for 15 min. The lysate was then transferred to a test tube. A total of 20 μL Passive Lysis Buffer lysis buffer was transferred into a white 96-well plate, then 100 μL of LARII (Promega) was added to detect Firefly luciferase in Multifunctional Enzyme Marker. A total of 100 μL of Stop&Glo (Promega) was used to detect Renilla luciferase activity.
5
miRNA and Tethering Assays in Mammalian Cells
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For miRNA assays, NIH3T3 cells were grown to 60–80% confluency on 24-well plates. Per well, 500 ng of FLAG-CSDE1 and its mutant constructs were transfected using JetPRIME reagent (Polyplus transfection) according to the manufacturer’s instructions. 24 h posttransfection, the cells were again transfected with 500 ng of the miRNA reporter constructs using the reagent. The cells were lysed 48 h posttransfection with 100 μl of 1× passive lysis buffer (Promega) and the reporter activity was measured using Luminoskan Ascent (Thermo Fisher Scientific). For tethering assays, HeLa and HEK293 cells were grown to 60–80% confluency on 24-well plates. Per well, 500 ng of λNHA-constructs were transfected using JetPRIME reagent (Polyplus transfection) according to the manufacturer’s instructions. 48 h posttransfection, the cells were again transfected with 50 ng (100 ng for experiments made with HEK293 cells) each of Renilla and Firefly luciferase reporter constructs using the reagent. The cells were lysed 24 h posttransfection with 100 μl of 1× passive lysis buffer (Promega), and the reporter activity was measured using Luminoskan Ascent.
6
GHK Peptide Effects on MSC Proliferation
7
Purification of Histones from Cells and Tumors
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Histones were purified as reported previously [34 (link)]. Briefly, cells (1–5 x 106) or lung tumors (10–100 mg) were lysated using a glass dounce in 1 ml of 1x Passive Lysis Buffer (Promega) and nuclear pellets were collected by centrifugation at 3,000 rpm for 5 min at 4 oC. The supernatant was discard and the pellets were washed with 1 ml of 1x Passive Lysis Buffer. Nuclear pellets were resuspended in 0.4 ml of the buffer (10 mM Trs-HCl, pH7.9, 0.2 mM EDTA, 1 mM DTT, 20% glycerol, 0.42 M KCl) and incubate for 30 min on ice, and centrifuged at 12,000 rpm for 10 min at 4 oC. The supernatant (histones extract) was transferred to a new tube and histones were precipitated by 20% trichloroacetic acid.
8
Luciferase Reporter Assay in HEK293 Cells
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HEK293 cells were seeded at a density of 5 x 104 cells per well in a 24-well plate 16 h prior to transfection. Cells were transfected for 16 h with 50 ng of luciferase reporter plasmid (AP-1 or NF-ĸB dependent luciferase reporter plasmid), 30 ng of pTK-Renilla luciferase, and 100 ng of expression vectors (myc-effector or myc vector alone). For MAPK reporter assays, cells were then incubated with 25 ng/ml of PMA for 6 h and harvested in 100 μl of passive lysis buffer (Promega). For NF-ĸB reporter assays, cells were then incubated with 10 ng/ml of TNF-α for 8 h and harvested in 100 μl of passive lysis buffer (Promega). Luciferase activity was measured using Dual Luciferase reporter assay system (Promega) and a TD20/20 Luminometer (Turner Designs) and normalised according to Renilla luciferase intensity. The data presented are from at least three independent experiments.
9
Metformin Modulates HIF1-α and NF-κB Activity
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For HIF1-α luciferase assays, RAW 264.7 cells were seeded at a concentration of 2.0 × 105 cells/ml and transfected with 1 µg pGL2-HRE-Luc (a gift from Navdeep Chandel; Addgene plasmid #26731) using 5 µg PEI Max (Polysciences) per ml95 (link). Beginning 24 h after transfection, cells were treated with 5 mM metformin or an equivalent volume of PBS for 6 h prior to stimulation with 100 ng/ml LPS for up to 24 h. Treatments were staggered to permit simultaneous harvest of all samples. Cells were washed in PBS and incubated overnight or longer at -80 °C in 300 μl of Passive Lysis Buffer (Promega). Luciferase activity in each lysate was then assessed using the Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer’s protocol. For NF-κB luciferase assays, RAW 264.7 cells containing the stably integrated pBIIx-Luc NF-κB reporter plasmid were seeded in duplicate at a concentration of 8.0 × 105 cells/ml and pretreated with 1 mM metformin for up to 24 h prior to stimulation with 100 ng/ml LPS for 2 h. Treatments were staggered to permit simultaneous harvest of all samples. Cells were washed in PBS and incubated overnight at -80 °C in 500 μl of Passive Lysis Buffer (Promega). Luciferase activity in each lysate was then assessed using the Dual-Luciferase Reporter Assay System (Promega) as per the manufacturer’s protocol. Results were normalized against untreated samples.
10
Quantifying Type I Interferon Levels
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Total amounts of IFN-α/β in cell supernatants were measured by using 293T cells stably expressing the firefly luciferase gene under the control of the mouse Mx1 promoter (Mx1-luc reporter cells) [76 (link)]. Briefly, cell supernatants were harvested and virus particles were removed with Amicon spin columns with a cutoff of 100 kDa (Millipore) according to the manufacturer's instructions. Mx1-luc reporter cells were seeded into 96-well plates in sextuplicates and were treated 24 hours later with filtered supernatants diluted 1:10 in DMEM-5% FCS. At 16 hours post incubation, cells were lysed in passive lysis buffer (Promega), and luminescence was measured with a microplate reader (Tecan). The assay sensitivity was determined by a standard curve.
For reporter assays, HEK293 cells were plated in 96-well plates 24 hours prior to transfection. Firefly reporter and Renilla transfection control were transfected using polyethylenimine (PEI, Polysciences) in sextuplicates for untreated and treated conditions. In 24 hours cells were stimulated for 16 hours with corresponding inducer and harvested in passive lysis buffer (Promega). Luminescence of Firefly and Renilla luciferases was measured using dual-luciferase-reporter assay (Promega) according to the manufacturer’s instructions in a microplate reader (Tecan).
For reporter assays, HEK293 cells were plated in 96-well plates 24 hours prior to transfection. Firefly reporter and Renilla transfection control were transfected using polyethylenimine (PEI, Polysciences) in sextuplicates for untreated and treated conditions. In 24 hours cells were stimulated for 16 hours with corresponding inducer and harvested in passive lysis buffer (Promega). Luminescence of Firefly and Renilla luciferases was measured using dual-luciferase-reporter assay (Promega) according to the manufacturer’s instructions in a microplate reader (Tecan).
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