CA-MSCs were encapsulated into 3% alginate as previously described (Schmitt et al., 2015 (link)). Briefly, 3% w/v sodium alginate was dissolved in PBS, filtered for sterility with a 0.2um filter. Cells were mixed with the alginate solution and added dropwise into a 5mM solution of calcium chloride to allow for gelation. Alginate beads were washed 2x with PBS prior to use in assays. Cell viability over time was verified by transection of the alginate capsule after 2 weeks incubation in standard culture media (DMEM, 10% FBS, 1% pen/strep) and re-isolation and growth of previously encapsulated CA-MSCs. Also, CA-MSCs lentivirally transduced with a secreted luciferase construct were encapsulated in alginate and the media was sampled for secreted luciferase overtime using the Ready-to-glow Secreted luciferase reporter system (Takara).
Ready to glow secreted luciferase reporter system
Manufactured by Takara Bio
Sourced in United States
The Ready‐To‐Glow Secreted Luciferase Reporter System is a lab equipment product that enables the detection and quantification of secreted luciferase activity in cell culture media or other biological samples. It provides a straightforward and sensitive method for monitoring gene expression or cellular events.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Psv β galactosidase vector, by Promega (1 mentions)
β galactosidase enzyme assay system, by Promega (1 mentions)
Fugene hd, by Promega (1 mentions)
Taq dna polymerase, by Roche (1 mentions)
Bovine serum albumin (bsa), by Promega (1 mentions)
T4 dna ligase, by Promega (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Seap reporter gene assay kit, by Roche (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
Infinite m200 luminometer, by Tecan (1 mentions)
Secrete pair gaussia luciferase assay kit, by GeneCopoeia (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Spectramax m5 luminometer, by Molecular Devices (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
8 protocols using ready to glow secreted luciferase reporter system
1
Alginate Encapsulation of CA-MSCs
2
Alginate Encapsulation of CA-MSCs
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CA-MSCs were encapsulated into 3% alginate as previously described (Schmitt et al., 2015 (link)). Briefly, 3% w/v sodium alginate was dissolved in PBS, filtered for sterility with a 0.2um filter. Cells were mixed with the alginate solution and added dropwise into a 5mM solution of calcium chloride to allow for gelation. Alginate beads were washed 2x with PBS prior to use in assays. Cell viability over time was verified by transection of the alginate capsule after 2 weeks incubation in standard culture media (DMEM, 10% FBS, 1% pen/strep) and re-isolation and growth of previously encapsulated CA-MSCs. Also, CA-MSCs lentivirally transduced with a secreted luciferase construct were encapsulated in alginate and the media was sampled for secreted luciferase overtime using the Ready-to-glow Secreted luciferase reporter system (Takara).
3
Transient Transfection and Reporter Assay
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KPC cells were transiently cotransfected with pNFκB‐Met‐Luc reporter plasmid (Clontech Laboratories, Mountain View, CA, USA) and internal control pSV‐β‐Galactosidase Vector (Promega Corp., Madison, WI, USA) by FugeneHD (Promega Corp.). After 48 hours, the medium was replaced with DMEM containing 10% FCS, collected 8 hours later, and luciferase activities were quantified using a Ready‐To‐Glow Secreted Luciferase Reporter System (Clontech Laboratories). Values were normalized relative to β‐galactosidase activity detected by β‐Galactosidase Enzyme Assay System (Promega Corp.).
4
Extraction and Characterization of Medicinal Plant Compounds
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We procured dried Glycyrrhiza uralensis and Platycodon grandiflorus roots, as well as dried plants without roots, from Andrographis paniculata, Centella asiatica, Gynostemma pentaphyllum, Polygonum chinense, Portulaca oleracea, Saururus chinensis, Smilax china, and Taraxacum campylodes from a reputable Chinese medicinal herb store in Taiwan (July 2020). Taq DNA polymerase was obtained from Kapa Biosystems (Roche, Basel, Switzerland). pMetLuc2 plasmid DNA and Ready-To-Glow™ Secreted Luciferase Reporter System were obtained from Clontech (Mountain View, CA, USA). Bovine serum albumin (BSA), T4 DNA ligase, and restriction enzymes (BglII and BamHI) were purchased from Promega (Madison, WI, USA). Gancaonin G and licoisoflavone A were obtained from ChemFaces (Wuhan, Hubei, China). Cudraflavone-C was purified in Dr. Liang’s laboratory (Figure S1 ). Methylthiazoletetrazolium (MTT), dimethyl sulfoxide (DMSO) and Geneticin™ selective antibiotic (G418 sulfate) were obtained from Sigma (St Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Ethanol was obtained from J. T. Baker (Phillipsburg, NJ, USA). In this study, all other chemicals and solvents utilized were of reagent or high-performance liquid chromatography (HPLC) quality.
5
Transient Transfection Reporter Assay
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HT1080 cells were cultured in DMEM/10% FCS. For reporter assays cells were plated at 5 × 104 cells/well in 12‐well plates overnight to reach 60–70% confluence. Cells were transfected by jetPEI (Polyplus, Strasbourg, France) with 0.6 µg/well of total DNA (pSEAP reporter construct and pMetLuc for normalization mixed at 1:20 molar ratio) in DMEM/0.3% FCS. Reporter activity was measured 24 hr after transfection. Alternatively, cells were transiently transfected in 3% FBS or 3% DCC‐treated FBS for 24 hr. Secreted alkaline phosphatase (SEAP) activity in the culture medium was determined using the SEAP Reporter Gene Assay Kit (Roche diagnostics) and for normalization the Ready‐To‐Glow secreted luciferase reporter system (Clontech) and measured in a luminometer (Mithras LB940; Berthold technologies, Bad Wildbad, Germany). SEAP values were normalized to Luciferase values to control for transfection efficiency. The normalized luminescence values were then standardized by the background activity (empty vector). The pSEAP‐TNW‐79 bp construct was included in all experiments and was used to calibrate different experiments. Error bars represent the standard error of the mean (SEM) between all replicates of each experiment.
6
Osteoblast Transcriptional Response to Galectin-8
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Osteoblasts were transfected with pNFκB-MetLuc2-Reporter, pMET-Luc-SDF-1 or with a control p-MET-Luc2 vector and were treated with gal-8 24 h post transfection. NFkB or SDF-1 promoter activity in response to gal-8 was determined using the “Ready-To-Glow secreted luciferase Reporter System” according to the manufacturer’s instructions (Clontech, Mountain View, CA, USA). Secreted luciferase levels were measured using the TECAN infiniteM200 luminometer.
7
Transient Transfection and Luciferase Assay
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HepG2 cells were seeded into 48-well plates and transiently transfected with a reporter gene construct (either pPXR1-Gluc or pPXR2-Gluc) together with expression vectors using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The next day, the medium was changed based on the experimental setting to either conditioned or nonconditioned Opti-MEM I Reduced Serum Medium supplemented with 5% FBS. After 24 h, medium was analyzed for activities of both SEAP, with Ready-To-Glow Secreted Luciferase Reporter Systems (Clontech, Palo Alto, CA, USA), and Gaussia luciferase, with Secrete-Pair Gaussia Luciferase Assay Kit (GeneCopoeia, Rockville, MD) and GL-S buffer for more stable activity. The Gaussia luciferase and SEAP signals were detected by the plate reader Synergy2 (BioTek, Winooski, VT), and Gaussia luciferase activities were normalized to SEAP activities. The data are presented as the relative change in normalized Gaussia luciferase activities to control activities, which were defined as 100%. The data are presented as the mean ± SD (n = 3).
8
NF-κB Promoter-Metridia Luciferase Assay
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The pNFκB-Metridia luciferase reporter plasmid contains an NF-κB promoter element upstream of the secreted Metridia luciferase (MetLuc) gene (Clontech Laboratories, Mountain View, CA). The pMetridia luciferase reporter plasmid contains several cloning sites that allow the insertion of promoter and/or enhancer elements upstream of the secreted MetLuc gene (Clontech Laboratories), which serves as an internal standard of transfection. Transient transfection was achieved using Lipofectamine® LTX (Life Technologies) according to the manufacturer’s instructions. Briefly, sub-confluent cells in 24-well plates were transiently transfected with 0.5 μg of the reporter construct and then stimulated 24 h later with whole P. gingivalis cells for 48 h in E-MEM containing 5% FBS. Secreted Metridia luciferase activity in culture supernatants was analyzed using Ready-To-Glow™ Secreted Luciferase Reporter Systems (Clontech Laboratories) and a SpectraMax M5 luminometer (Molecular Devices, Menlo Park, CA).
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