HEK293 cells were plated in 96-well plates at a density of 4×104 cells per well, and transfected the following day with plasmids bearing firefly luciferase, Renilla luciferase, recombinant murine TLR4 and MD-2 or recombinant murine TLR2 and TLR1 by standard calcium phosphate precipitation. After overnight transfection, the test wells were stimulated in triplicate for 4 hours at 37°C with the indicated doses of LPS isolates or live bacteria. Following stimulation, the transfected HEK293 cells were rinsed with phosphate-buffered saline and lysed with 50 µl of passive lysis buffer (Promega, Madison, WI). Luciferase activity was measured using the Dual Luciferase Assay Reporter System (Promega, Madison, WI). Data are expressed as fold increase of NF-κB-activity which represents the ratio of NF-κB-dependent fire-fly luciferase activity to β-Actin promoter-dependent Renilla luciferase activity.
Phosphate buffered saline (pbs)
Manufactured by Promega
Sourced in United States
PBS is a buffer solution commonly used in various laboratory applications. It is a balanced salt solution that maintains a neutral pH and osmotic environment, making it suitable for a wide range of biological and biochemical procedures. The core function of PBS is to provide a physiologically compatible medium for the handling and processing of cells, tissues, and other biological samples.
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Lab products found in correlation
D luciferin, by Promega (2 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Luciferin, by Promega (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (1 mentions)
Trisodium citrate dihydrate, by Merck Group (1 mentions)
Gold 3 chloride trihydrate, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Promega (1 mentions)
Celltiter96 aqueous one solution reagent mts compound, by Promega (1 mentions)
Hs peg cooh, by Merck Group (1 mentions)
Mem eagle, by Sartorius (1 mentions)
Herceptin, by Roche (1 mentions)
Lysis buffer, by Promega (1 mentions)
Ivis spectrum system, by PerkinElmer (1 mentions)
Td 20 20 luminometer, by Promega (1 mentions)
Isoflurane, by Sumitomo Pharma (1 mentions)
Luciferase assay system, by Promega (1 mentions)
9 protocols using phosphate buffered saline (pbs)
1
NF-κB Activation Assay in HEK293 Cells
2
Knockdown of circRNA_002144 Inhibits Tumor Growth and Metastasis
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Animal study was approved by the Ethics Committee of Hainan Affiliated Hospital of Hainan Medical University and in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines. Twelve 5-week-old female BALB/c nude mice (20–25 g) were randomly divided into two groups (n = 6). T84 cells (5 × 106/0.1 mL PBS) with stable knockdown of hsa_circRNA_002144 or shNC were subcutaneously injected into right flank of nude mice. Ten days later, tumor volume was calculated every two days. Twenty days later, the mice were killed with 40 mg/kg sodium pentobarbital, the tumor tissues were isolated, weighed and photographed. For detection of metastasis, the T84 cells were injected into NOD/SCID mice by tail vein. After intraperitoneal injection of luciferin (4 mg/50 μL phosphate buffered saline; Promega) for 10 min, the metastases were photographed by IVIS Lumina II system (Caliper LifeSciences, Hopkinton, MA, USA). Lung tissues were also isolated, and fixed in 10% formalin and embedded in paraffin. The sectioned lung tissues were subjected to H & E staining, and the representative images were observed via microscope (Olympus).
3
Conjugation of Trastuzumab to Gold Nanoparticles
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The following chemical reagents were used: gold (III) chloride trihydrate (HAuCl4·3H2O), trisodium citrate dihydrate (C6H9Na3O9), HS–PEG–COOH (poly(ethylene glycol), 5 kDa) from Sigma-Aldrich (St. Louis, MO, USA), and OPSS–PEG–NHS (alpha-pyridyl-2-disulfid-omega-carboxy succinimidyl ester poly(ethylene glycol), 5 kDa) from Creative PEGworks (Chapel Hill, NC, USA). Trastuzumab was isolated from Herceptin (Roche Pharmaceuticals, Basel, Switzerland). Iodogen (1,3,4,6-tetrachloro-3R,6R-diphenylglycouril) from Thermo Fischer Scientific (Waltham, MA, USA), and PD-10 column (GE Healthcare, Piscataway, NJ, USA). Hydrochloric acid and sodium hydroxide were purchased from POCH (Gliwice, Poland). Fluorescence mounting medium was obtained from Dako (Carpinteria, CA, USA). The following materials were also used in cell studies: McCoy’s, DMEM, MEM-EAGLE, and RPMI mediums and fetal calf serum from Biological Industries (Beth Haemek, Israel), phosphate-buffered saline (PBS), dimethylsulfoxide (DMSO), and the CellTiter 96® Aqueous One Solution Reagent (MTS compound) from Promega (Mannheim, Germany). SKOV-3 and MDA-MB231 cells were also purchased from the American Type Tissue Culture Collection (ATCC, Rockville, MD, USA) and maintained based on the ATCC protocol. Additionally, all solutions were prepared using double-distilled water (18.2 MΩ·cm, Hydrolab, Straszyn, Poland).
4
Measuring Angiogenesis Biomarkers in Serum
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To measure uPA, tPA, and PAI-1 antigen levels, venous blood was collected into plastic tubes containing 3.8% sodium citrate (ratio 9:1). The samples were centrifuged at 1,500 g for 15 min at Ϫ4 °C, and the supernatant was stored at Ϫ80 °C until it was examined. Aliquots of the appropriately diluted samples were assayed by enzymelinked immunosorbent assay following the instructions of the manufacturer (PAI-1/tPA and uPA IMUBIND ELISA kits, American Diagnostica, Pfungstadt, Germany).
Active TGF- was assayed by adding 100 l of serum for 16 h to subconfluent mink lung epithelial cells that were stably transfected with a PAI-1 promoter luciferase construct. 26 At the end of culture, the cells were washed twice with phosphate-buffered saline and lysed in 60 l of lysis buffer (Promega, Madison, Wisconsin). The cell lysates were assayed for luciferase activity with an assay kit from Promega using a Berthold luminometer. Parallel mink lung epithelial cells were incubated with known increasing concentrations of TGF- to obtain a standard curve.
Active TGF- was assayed by adding 100 l of serum for 16 h to subconfluent mink lung epithelial cells that were stably transfected with a PAI-1 promoter luciferase construct. 26 At the end of culture, the cells were washed twice with phosphate-buffered saline and lysed in 60 l of lysis buffer (Promega, Madison, Wisconsin). The cell lysates were assayed for luciferase activity with an assay kit from Promega using a Berthold luminometer. Parallel mink lung epithelial cells were incubated with known increasing concentrations of TGF- to obtain a standard curve.
5
Luciferase Imaging of Thigh Muscle
6
NF-κB Activation Measurement in RAW Cells
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NF-κB activation was checked using a luciferase reporter plasmid, pTAL-NF-κB (Stratagene, La Jolla, CA). Transfections were carried out using Amaxa Nucleofector Technology. Briefly, RAW 264.7 cells were passaged 2 days before nucleofection, on the day of the experiment 100 µL of a cell suspension at 0.5×106 cell/mL was combined with 3 µg of highly purified DNA plasmid, transfected according to the manufacturer protocol and incubated for 12 h in a 5% CO2 atmosphere at 37 °C. Thereafter, cells were stimulated with LPS (1 µg/mL), either in the absence or in the presence of indicaxanthin (50, 75, 100 µM). After 6 h cells were washed with PBS and lysed for 5 min at 4 °C according to the manufacturer’s instructions (Promega, Madison, WI). Luciferase activity is expressed as relative luminescence units (RLU) x103 following reading in a TD/2020 luminometer (Turner Biosystems, Sunnyvale, CA).
7
Resuscitation of Ischemic Rat Liver
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Donor rats were euthanised by cervical dislocation and underwent 90-min cardiac arrest at room temperature. Then, an ischaemic liver was isolated and connected to our perfusion culture system in the same manner as a fresh liver. In the 100-min perfusion culture for the ischaemic liver, the ALT concentration of the effluent was evaluated as an indicator of organ damage. We also analysed the amount of internal ATP in the resurrected liver using the non-invasive bio-imaging system IVIS (Xenogen, Alameda, CA, USA) and the IVIS Living Image (Xenogen) software package. Luc-Tg rats were treated by ischaemic conditioning, and the livers of the perfusion circuit were established in the same manner. During the resuscitative procedure, we injected 3 mL of D-luciferin dissolved and diluted to 15 mg/mL in PBS (Promega, Madison, WI, USA) into the perfusate directly before the liver to detect the luminescence from the Luc-Tg liver. The signal intensity was quantified as photon flux in units of photons/s/cm2 of a steradian in the region of interest. After the resuscitative procedure by perfusion culture, these livers were transplanted to the recipient rats using the liver auxiliary transplantation method.
8
Bioluminescence Imaging of Toxoplasma Infection
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Female BALB/c mice were used in experiments. Mice were intraperitoneally infected with 103 freshly egressed tachyzoites expressing luciferase resuspended in 100 µl PBS. For the local infection, mice were infected in the right hind footpad with 103 freshly egressed tachyzoites expressing luciferase resuspended in 50 µl PBS. Bioluminescence was assessed on the indicated days after infection. For the detection of bioluminescence emission, mice were intraperitoneally injected with 3 mg d -luciferin in 200 µl PBS (Promega), maintained for 5 min to allow for adequate dissemination of luciferin, and subsequently anaesthetized with isoflurane (Dainippon Sumitomo Pharma). At 10 min after injection of d -luciferin, photonic emissions were detected using an in vivo imaging system (IVIS-Spectrum; Xenogen) and Living image software (Xenogen).
9
Luciferase Reporter Plasmid Transfection
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The luciferase reporter plasmids (vpHW72-Luc and canine pHW72-Luc), which contained the Vero cell-and MDCK celldriven polI transcription unit, respectively, were generated in the backbone of vpHW72-Luc, as previously described [17, 20] . Briefly, Vero or MDCK cells were cultured in 24 h prior to use and 0.1 μg each of the vpHW72-Luc or canine pHW72-Luc, vpHW2000-PB2, vpHW2000-PB1, vpHW2000-PA, vpHW2000-NP, and pCMV-βgalactosidase plasmids were transfected into each cells with TransIT-LT1 transfection reagent, as previously described [16] . Fresh DMEM (Gibco, USA) containing 5% FBS and incubated at 37 o C was used to replace the transfection medium after 4 h. Luciferase activity was assayed in triplicate using the luciferase assay system with each of the cell lysates subsequent to washing with PBS after 24 h (Promega, USA). The results were normalized to the β-galactosidase activity level of the cells.
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