The GloSensor cAMP assay was used to measure activating effects of HCAR2 and its mutations referring to the previous research.63 (link) Firstly, pcDNA3.1-HCAR2 or its mutations with GloSensor reporter plasmids were co-transfected into a six-well plates confluent with HEK293 cells. After 24 h transfection, the cells were re-plated in 96-well plates with the Hank’s Balanced Salt Solution (HBSS) buffer containing d -Luciferin-Potassium Salt (YEASEN). Then the cells were stimulated with the corresponding agonist with adding 5 μM forskolin at the same time. To determine the allosteric regulation efficacy, different concentrations of compound 9n were added additionally. Finally, Synergy H1 microplate reader (BioTek) was used to read the luminescence. Data were fitted in GraphPad Prism 9 by using the nonlinear regression (curve fit) dose-response function.
D luciferin potassium salt
Manufactured by Yeasen
Sourced in China
D-luciferin potassium salt is a chemical compound that serves as a substrate for the bioluminescent enzyme luciferase. It is commonly used in various analytical and experimental applications, particularly in the field of cell biology and biomedical research.
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Lab products found in correlation
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Mitochondrial membrane potential detection kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Cell culture medium, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8 assay kit, by MedChemExpress (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cdk7 inhibitor thz1, by MedChemExpress (1 mentions)
Enhanced chemiluminescence ecl kit, by PerkinElmer (1 mentions)
Fluo 4 am, by Yeasen (1 mentions)
Anti calreticulin, by Santa Cruz Biotechnology (1 mentions)
Fitc labeled cd11c, by BioLegend (1 mentions)
Pe labeled cd8a, by BioLegend (1 mentions)
Apc labeled cd4, by BioLegend (1 mentions)
Fetal bovine serum albumin fbs, by Biowest (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Hmgb1, by Santa Cruz Biotechnology (1 mentions)
Ivis lumina lt series 3, by PerkinElmer (1 mentions)
In fusion kit, by Takara Bio (1 mentions)
Ivis spectrum, by PerkinElmer (1 mentions)
11 protocols using d luciferin potassium salt
1
GloSensor cAMP Assay for HCAR2 Activation
2
In utero electroporation for brain tumor modeling
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The in utero electroporation-operated embryos were allowed to survive until P21–P30 and were subjected to bioluminescence imaging of Luc activity. Luc signals were captured using the IVIS Lumina II (Caliper Life Sciences). Mice were injected with 30 mg of D-Luciferin, Potassium Salt (Yeasen Biotech) intraperitoneally 8 min before imaging. Images were acquired and radiance was determined within mouse heads. Only mice whose brain carried Luc signals were selected for further analysis. At postnatal days 60–150, all selected mice were subjected to bioluminescence imaging again to make sure tumors were big enough for experiments.
3
Bioluminescent Tumor Tracking in Mice
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To observe tumor growth in living mice, luciferase images were obtained using the Small Animal In Vivo Imager system to track luciferase expressed by tumor cells. D-Luciferin Potassium Salt (Yeasen, Shanghai, China) was injected intraperitoneally into mice at a concentration of 150 milligram/kilogram (luciferin/body weight) 10 min before imaging and the Living image version 4.2 was used to quantify the expression of luciferase.
4
Characterization of Apoptosis Induction
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Calcium chloride dihydrate (CaCl2·2H2O), alendronate sodium trihydrate (NaALN·3H2O) were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China). CDK7 inhibitor THZ1 and the cell counting kit-8 (CCK8) assay kit were purchased from MedChemExpress (MCE, Shanghai). Dimethyl sulfoxide (DMSO) and penicillin–streptomycin (pen/strep) solution (100 ×) were purchased from Sigma Aldrich (MO, USA). Cell culture medium, trypsin–EDTA and fetal bovine serum (FBS) were provided by Gibco (Guangzhou, China). Annexin V-FITC/PI apoptosis detection kits and mitochondrial membrane potential detection kit were purchased from Beyotime (Shanghai, China). 6-carboxy fluorescein (6-FAM), DAPI dihydrochloride and DiR iodide were purchased from Invitrogen (Carlsbad, CA, USA). D-Luciferin potassium salt and Fluo-4, AM were acquired from Yeasen Biotech Co., Ltd (Shanghai, China). Acridine orange-ethidium bromide (AO/EB) staining kit was purchased from Leagene Biotechnology (Beijing, China). Bcl-2, Bax and cleaved caspase-3 antibodies were bought from Absin Bioscience (Shanghai, China). β-actin and secondary antibodies were purchased from Proteintech (Wuhan, China). BCA protein quantitation kit and enhanced chemiluminescence (ECL) kit were purchased from Perkin-Elmer (Waltham, USA). All other reagents were consistent with previous report [35 (link)].
5
Comprehensive Immunomodulatory Assay Protocol
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Thymopentin (TP5) was purchased from Meilunbio. Doxorubicin hydrochloride (DOX) was obtained from Aladdin. Fetal bovine serum albumin (FBS) was purchased from Biowest. Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco. All assay kits, if not specified, were acquired from Beyotime. The antibodies used were as follows: anti-calreticulin, HMGB1, and CD8 were purchased from Santa Cruz Biotechnology; Ki67 was purchased from Bioss; anti-mouse antibodies (FITC-labeled CD3, FITC-labeled CD11c, FITC-labeled CD25, PE-labeled CD8a, PE-labeled CD86, APC-labeled CD4, APC-labeled CD80, Alexa Flour 647-labeled Foxp3) were all purchased from BioLegend. D-Luciferin, Potassium Salt was purchased from Yeasen.
6
Bioluminescent Imaging of Tumor Burden
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To monitor tumor burden, mice were intraperitoneally injected with 150 mg/kg D-luciferin potassium salt (40902ES02, Yeasen). After 5 mins, mice were anesthetized with isoflurane and imaged by the IVIS® Spectrum Imaging System. The bioluminescent signal was measured in the region of interest to estimate tumor burden. Detailed are described in supplemental materials.
7
Dual-Luciferase Transient Expression Assay
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The RLM1 CDS was cloned into pCAMBIA1300-nLUC by an In-Fusion kit (Clontech, Japan), and the OsMAPK10 CDS was cloned into pCAMBIA1300-cLUC. The plasmid was sequenced for verification and then transformed into Agrobacterium strain GV3101. Four-week-old N. benthamiana plants were infiltrated by the transformed Agrobacterium cells and incubated at 23°C under dark conditions for 2 days; pCAMBIA1300-nLUC with pCAMBIA1330-cLUC served as a blank control. The leaves were sprayed with D-luciferin potassium salt (Yeasen, Shanghai, China), and the fluorescence was detected by an IVIS Lumina LT series III instrument (PerkinElmer).
8
Metastatic Lung Tumor Model in Nude Mice
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Four-week-old male athymic BALB/c nude mice were purchased from Vital River Laboratory (Beijing, China). All protocols for animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Zhengzhou University. The stable cell lines of HCT116-LV-NC and HCT116-LV-CircTUBGCP4 (2 × 106 in 100 µL of PBS) were injected via the tail vein. After 30 days and 40 days, the mice were injected intraperitoneally 100 μl D-luciferin potassium salt (Yeasen, Shanghai, China). Then In vivo imaging was acquired with the IVIS Spectrum (PerkinElmer, Waltham, Massachusetts, USA). After 40 days, mice were sacrificed and lungs were removed following HE staining and IHC.
9
In Vivo Bioluminescence Imaging of Tumor Cells
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Four- to five-week-old BALB/c or BALB/c-nude female mice were used in our experiments. For in vivo imaging technology, 2–5 × 106 cells carrying Luciferase (LUC) coding sequences were injected into mice. After 10–15 days, D-Luciferin potassium salt (150 mg/kg) (40902ES02, YEASEN, China) was injected into the abdominal cavity of anesthetized mice, the latter were placed in the Chemiluminescence Gel Imaging System Tanon-5200Multi (Shanghai Tanon, China) to observe live imaging.
10
Bioluminescent Mouse Model of Metastatic Breast Cancer
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A group of 4-week-old female BALB/c nude mice were purchased from the Animal Core Facility of Nanjing Medical University. Cell line-derived xenograft (CDX) mouse models were used in this study. The number of animals used for each experiment is indicated in each figure or figure legend. Prior to carrying out the experiment, mice were randomly assigned to different treatment groups. 1×105 MDA-MB-231-luc cells (MDA-MB-231 cells labeled with luciferase tag) were suspended in 100 μl PBS and injected into nude mice through the caudal vein. Thereafter, luciferase signals in the mice were measured every 2 weeks. For imaging, the mice were injected intraperitoneally with D-Luciferin Potassium Salt (Yeasen, Shanghai, China) at 150 mg/kg of body weight and imaged in the IVIS system (PerkinElmer, USA) after 10 min while anesthetized. On the 45th day of the experiment, the mice were sacrificed and subsequently dissected. Their lungs were collected for observation in vitro and fixed with 4% paraformaldehyde for subsequent experiments, such as hematoxylin-eosin (HE) and IHC staining. A blinding strategy when assessing the outcome was used whenever possible.
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