DNA and glycosaminoglycan (GAG) content were measured for discs in the Control and Decellularized groups to characterize the effect of SDS treatment. Each disc was digested overnight at 60 °C in 1 ml of papain digestion buffer (100 mM sodium phosphate buffer, 10 mM Na2EDTA, 10 mM L-cysteine, and 0.125 mg/mL papain, pH 6.5). An aliquot of the digestate was mixed with Hoechst assay buffer containing 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 0.2 μg/ml bisBenzimide H 33258. Fluorescence (excitation 365 nm, emission 410 – 450 nm) was read using a multimode reader (GloMax®-Multi Jr, Promega, Madison, WI). DNA content was determined by comparison to a standard curve generated from calf thymus DNA. A separate aliquot of the digestate was taken for determination of GAG content using a dimethylmethylene blue assay (Blyscan™ Assay, Biocolor Ltd, Carrickfergus, UK). The assay was performed according to the manufacturer’s recommended protocol.
Glomax multi jr
Manufactured by Promega
Sourced in United States
The GloMax-Multi Jr. is a multi-mode microplate reader designed for a variety of luminescent, fluorescent, and absorbance-based assays. It provides accurate and reliable measurements across multiple detection modes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Caspase glo 3 7 assay, by Promega (2 mentions)
Dharmafect 1 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Pmirglo dual luciferase vector, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Blyscan assay, by Biocolor (1 mentions)
Prl tk vector, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase system, by Promega (1 mentions)
Prism 5, by GraphPad (1 mentions)
Ac devd amc, by Enzo Life Sciences (1 mentions)
Ac devd amc, by Merck Group (1 mentions)
Ac devd amc, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system kit, by Promega (1 mentions)
Dual luc reporter assay system, by Promega (1 mentions)
E1910, by Promega (1 mentions)
22 protocols using glomax multi jr
1
Characterizing Disc Decellularization Effects
2
Caspase-3/-7 Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells were treated with ACA (20 μM for Ca Ski and 30 μM for SiHa) for 6 h (plasmid transfection) or 12 h (miRNA transfection). The Caspase-Glo® 3/7 Assay (Promega, USA) was used to measure caspase-3 and -7 activities using GLOMAX®-Multi Jr (Promega, USA) according to the manufacturer’s protocol.
3
Characterization of PD-L1 Promoter Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The promoter sequence of human PD-L1 gene was obtained from the NCBI Gene database (Gene ID: 29126). A 922 bp PD-L1 promoter sequence starting from 872 bp upstream of the PD-L1 transcription start site (TSS) to 50 bp downstream of PD-L1 TSS (−872/+50) was cloned from the genome DNA (gDNA) of A549 cells, and further subcloned into pGL3-Basic Vector (Promega) to generate luciferase reporter plasmid (pGL3-WT). The potential NRF2 binding site sequence (5′-ATGACAAAGCA-3′, −593/–583) was analyzed by the JASPAR database (http://jaspar.genereg.net ), and the original binding site sequence was replaced by 5′-AAAAAAAAAAA-3′ to generate mutated luciferase reporter plasmid (pGL3-Mutant). All transfections for dual-luciferase reporter assay were performed by Lipofectamine 2000 (Invitrogen), and pRL-TK Vector (Promega) was used as transfection control. The luciferase activities in cell lysates were measured by GloMax Multi Jr (Promega) according to the manufacturer’s protocol.
4
Dual-Luciferase Assay for TEAD Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Dual-Luciferase Reporter Assay System (E1910, Promega, United States) was used to detect Luciferase activity. The cells were transfected with the TEAD luciferase reporter or pGL4.26-YAP–TEAD binding site and the Renilla plasmid. After 24 hours, cells were lysis, and luciferase activity was detected. A GloMax-Multi Jr. (Promega-GloMax Promega, USA) was used to detect luciferase activity.
5
Luciferase Assay for miRNA Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were co-transfected with 40 ng of 3′UTR reporter constructs containing wild-type or mutated binding sites and 100 nM of miR-210 mimic or mimic negative control using DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific, USA). The firefly and Renilla luciferase activities were measured 48 h post-transfection with Dual-Glo® Luciferase Assay System (Promega, USA) according to the manufacturer’s protocol using GLOMAX®-Multi Jr (Promega, USA). The firefly luciferase activity was normalized to Renilla activity, which was used as an internal control.
6
Intracellular ATP Quantification in Epimastigotes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular ATP (ATPi) was measured using an ATP bioluminescent somatic cell test kit (Sigma-Aldrich). In brief, epimastigotes (1 × 107 parasites per tube) were incubated in a solution containing 100 mM sucrose, 50 mM KCl, and 50 mM Tris-HCl in 0.1 mL (pH 7.2 adjusted with HCl). Cellular extracts were prepared by combining them with 0.1 mL of somatic cell ATP-releasing reagent and then chilling the mixture for 1 min. The mixture was transferred to MTS-11C mini tubes containing 0.1 mL ATP assay mix (v:v; Axygen) and swirled for 10 s at room temperature. A GloMax Multi JR detection system (Promega) measured the overall quantity of light emitted. In each experiment, the total intracellular ATP concentration per 107 cells was determined using a standard ATP curve [29 (link)].
7
Luciferase Assay for circFBXW4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
circFBXW4 sequences contain the target sites for miRNA candidates were synthesized and cloned into pSI-Check2 reporter vector downstream to firefly luciferase (pSI-Check2-circFBXW4-wildtype), mutant version of circFBXW4 (pSI-Check2-circFBXW4-mutant) was also generated with the deletion of complementary sites, respectively. The reporter vector, miR-18b-3p mimics or negative control were co-transfected in HEK-293T cells using Lipofectamine 3000 (Invitrogen, CA). Activity of firefly and renilla luciferase were measured by Dual-Luciferase system (Promega, USA) according to the manufacturer's protocol and detected by GloMax Multi Jr (Promega, USA).
8
Luciferase Assay for miRNA Targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cotransfected with 40 ng of 3′-UTR reporter constructs containing wild-type or mutated binding sites and 100 nM of miR-629 mimic or negative control using DharmaFECT 1 Transfection Reagent (Thermo Fisher Scientific). Firefly and Renilla luciferase activities were assayed 48 h after transfection with Dual-Glo® Luciferase Assay System (Promega) according to the manufacturer’s protocol using GloMax-Multi Jr (Promega). The firefly luciferase activity was normalized to Renilla activity, which was used as an internal control.
9
Luciferase Assay for miR-146a Target
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 996-bp segment of the human EGR1 3′UTR containing the miR-146a site was cloned into the pmiRGlo dual luciferase vector (Promega). A similar cloning strategy was used to clone murine Egr1 3′UTR and the Nrp2 UTR (see Supplementary Table 1 ). For mutation of the miR-146a binding site, we utilized site-directed mutagenesis as previously described using the primers shown in Supplementary Table 1 [43 (link)]. Co-transfections were performed with Lipofectamine 2000 (Life Technologies) as per the manufacturer's instructions. Cells were lysed after 24 hours, substrate was added and luminescence was measured on a Glomax-Multi Jr (Promega).
10
Dual-Luciferase Reporter Assay for BmN Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reporter activity was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, BmN cells were lysed for 15 min at room temperature using 1× passive lysis buffer which is made from the dilution of the 5 × passive lysis (Promega, Madison, WI, USA), and then the lysed cells were collected and centrifuged at 12,000 g for 10 min. The supernatant was used for the determination of luciferase activity. Luciferase activity was measured using GloMax Multi Jr. at 490 nm (Promega, Madison, WI, USA). T-test was performed for statistical analysis by the software Graphpad Prism5.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!