The luciferase immunoprecipitation systems (LIPS) assay provides an informative tool to explore serology as evidence of autoimmunity and infectious disease exposure due to its ability to efficiently detect antigenicity antibodies against both conformational and linear epitopes. Here, LIPS was used to assess for the presence of autoantibodies against a small diverse panel of known and potential antigens in the PI-ME/CFS and healthy volunteer participants. The previously described testing format was used to examine antibodies against the various target molecules included known autoimmune-associated proteins (Ro52, Jo-1, TPO, gastric ATPase, tyrosine hydroxylase), neurological autoantigens (GAD65, LGI1, NMDAR1, MUSK), cytokines (Interferon alpha1, Interleukin-6, CXCR4, TGFB1), muscle proteins (MPZ, PMP22), as well as against several infectious agents (HDV, HEV, Zika virus). Light units were measured in a Berthold LB 960 Centro luminometer (Berthold Technologies, Germany) using coelenterazine or furimazine substrate mix (Promega, Madison, WI). In some cases, control sera samples from known positive control autoimmune patients were used as positive controls.
Centro lb 960 luminometer
Manufactured by Berthold Technologies
Sourced in Germany, United States, France
The Centro LB 960 is a luminometer, a laboratory instrument used to measure and quantify the amount of light emitted by a sample. It is designed to detect and analyze luminescent signals, which can be used in various applications such as bioluminescence and chemiluminescence assays.
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Lab products found in correlation
Dual luciferase reporter assay system, by Promega (13 mentions)
Passive lysis buffer (plb), by Promega (11 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Bright glo luciferase assay, by Promega (5 mentions)
Celltiter glo luminescent assay, by Promega (5 mentions)
Celltiter glo luminescent cell viability assay, by Promega (4 mentions)
Luciferase assay system, by Promega (3 mentions)
Dual light assay system, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Lsrfortessa, by BD (2 mentions)
Coelenterazine, by Carl Roth (2 mentions)
Maxiprep kit, by Qiagen (2 mentions)
Dual luciferase assay, by Promega (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Prl tk, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (2 mentions)
Apc brdu flow kit, by BD (2 mentions)
Dual luciferase assay system, by Promega (2 mentions)
96 well white microplate, by Thermo Fisher Scientific (2 mentions)
Renilla luciferase assay system, by Promega (2 mentions)
96 protocols using centro lb 960 luminometer
1
Comprehensive LIPS Assay for Autoantibody and Infection Detection
2
STAT3 Activation Assay Protocol
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The constitutively active STAT3C lentiviral vector (plasmid #24983) was obtained from Addgene (Cambridge, MA, USA). Plasmid expressing HA-tagged full-length human CDC6 was a gift from L. Drury (Clare Hall Laboratories, Cancer Research UK, London, England)30 (link). Luciferase reporter plasmid containing STAT3-responsive elements was obtained from Beyotime (Haimen, China). For luciferase assays, 0.2 µg STAT3 reporter construct was cotransfected with 0.02 µg of pRL-TK vector that provides constitutive expression of Renilla luciferase serving as an internal control. Six hours after transfection, cells were treated with or without BBR for another 24 h and the luciferase assays were performed using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA) and measuring with an LB960 Centro luminometer (Berthold Technologies, Germany). Transfections were performed in three independent experiments and assayed in quadruplicates. Data represent mean ± SD.
3
Wnt/β-Catenin Transcriptional Activation Assay
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β-Catenin–mediated transcription activation was determined using mouse L cells stably expressing SuperTOPFlash (firefly luciferase reporter driven by 7× TCF/LEF β-catenin binding sites) and β-galactosidase for normalization. Cells were seeded at 5 × 104 cells per well in 96-well culture plates, incubated overnight with Wnt3a or CHIR99021 at specified concentrations, then lysed in passive lysis buffer (Promega, Madison, WI) for reporter assay. For experiments with MDC, L cells were incubated with MDC with Wnt3a or CHIR99021 for 6 h at specified concentrations. Relative luciferase activity units were measured and normalized against β-galactosidase activity using the Dual-Light assay system (Thermo Fisher) on an LB 960 Centro luminometer (Berthold Technologies, Oak Ridge, TN). All assays were performed in triplicate.
4
SLE Autoantibody Detection with LIPS
5
Gaussia Luciferase Activity Assay
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Gaussia princeps luciferase activity was assayed using the BioLux Gaussia luciferase assay kit (New England Biolabs). Cleared spent culture medium was diluted 1:20 in 1× phosphate-buffered saline (PBS). A 2-μl aliquot of this solution was transferred to a 96-well black-bottom microwell plate (BD Biosciences, Franklin Lakes, NJ) containing 50 μl 1× PBS. GLuc assay solution (50 μl/well) was added by an injector-equipped LB960 Centro luminometer (Berthold Technologies, Bad Wildbad, Germany) at low speed. After a delay time of 1 s, the light emission corresponding to the GLuc enzymatic activity was measured (0.5-s counting time) in relative light units (RLU).
6
Dual-Luciferase Assay of ACBD3 Interaction
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HEK293T cells grown in 96-well plates were co-transfected with 50 ng of each pACT, pBIND, and pG5Luc plasmids using Fugene6 (Promega). At 24 hours post transfection, the cells were lysed, and both firefly and Renilla luciferase activities were measured using the Dual-Luciferase assay kit (Promega) and Centro LB 960 luminometer (Berthold Technologies) according to the manufacturer's instructions. The firefly luciferase activity was normalized to the Renilla luciferase activity (used as an internal control of the transfection efficiency) and then to the activity determined in cells co-expressing wild-type ACBD3 and 3A (which was set to 100%).
7
HCV Spreading and Replication Assays
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For HCV spreading experiments, lentiviral-transduced cells were analyzed using flow cytometry or Gaussia luciferase assays at the indicated timepoints as described before (17 (link), 75 (link)). Cells were either infected with Jc1NS5AB-EGFP (53 (link)) and analyzed by flow cytometry with a BD LSR Fortessa (BD Bioscience) and analyzed with FlowJo (Treestar) or infected with Jc1p7-GLuc-2A-NS2 (17 (link)), and Gaussia luciferase activity in the supernatant was measured using Coelenterazine (Carl Roth) and a Centro LB 960 luminometer (Berthold Technologies).
To measure HCV RNA replication, lentiviral-transduced cells were electroporated with Jc1ΔE1/E2NS5AB-FLuc (53 (link)) as described in (17 (link)) or JFH1FLuc-P2A-NS3-NS5B RNA, and Firefly luciferase activity in cell lysates was determined using Luciferase Assay System (Promega). Protein levels were determined by Coomassie-Plus Assay (Thermo Fisher Scientific).
To measure HCV RNA replication, lentiviral-transduced cells were electroporated with Jc1ΔE1/E2NS5AB-FLuc (53 (link)) as described in (17 (link)) or JFH1FLuc-P2A-NS3-NS5B RNA, and Firefly luciferase activity in cell lysates was determined using Luciferase Assay System (Promega). Protein levels were determined by Coomassie-Plus Assay (Thermo Fisher Scientific).
8
Characterizing Hepatocyte Metabolic Status
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The ATP concentration of hepatocytes was measured using an ATP assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's protocol. Briefly, the cells were lysed in lysis buffer and centrifuged at 4°C and 12 000 g for 10 minutes. The supernatants were used for ATP detection using a Centro LB960 luminometer (Berthold Technologies, Bad Wildbad, Germany). The concentration of ATP was calculated in accordance with a standard curve and converted into nmol/mg protein.
For the quantitative estimation of mitochondrial membrane potential, hepatocytes were incubated with 10 μmol/L rhodamine 123 (Beyotime Institute of Biotechnology) for 30 minutes in the dark, and the fluorescence intensity of the dye was determined by flow cytometry (Becton‐Dickinson, Mountain View, USA).
The intracellular ROS concentrations were measured using the peroxide‐sensitive fluorescent probe 2′7′‐dichlorofluorescein diacetate (DCFH‐DA) (Beyotime Biotechnology Inc., Nantong, China). The cells were exposed to serum‐free medium containing 10 μmol/L DCFH‐DA and propidium iodide in the dark for 30 minutes and then washed three times with cold PBS. The fluorescence was measured by flow cytometry (Becton‐Dickinson, Mountain View, USA).
For the quantitative estimation of mitochondrial membrane potential, hepatocytes were incubated with 10 μmol/L rhodamine 123 (Beyotime Institute of Biotechnology) for 30 minutes in the dark, and the fluorescence intensity of the dye was determined by flow cytometry (Becton‐Dickinson, Mountain View, USA).
The intracellular ROS concentrations were measured using the peroxide‐sensitive fluorescent probe 2′7′‐dichlorofluorescein diacetate (DCFH‐DA) (Beyotime Biotechnology Inc., Nantong, China). The cells were exposed to serum‐free medium containing 10 μmol/L DCFH‐DA and propidium iodide in the dark for 30 minutes and then washed three times with cold PBS. The fluorescence was measured by flow cytometry (Becton‐Dickinson, Mountain View, USA).
9
Optimizing Lentiviral Transduction Efficiency
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On the day before transduction, 60,000 293T cells were seeded in 96-well plate. The day of transduction, test samples of MS2RLP were applied to cells with or without 3 µg/ml puromycin. This translation inhibitor was added 1 hour before transduction. For 4070A envelopes transduction was realized in presence of Polybrene (5 µg/ml, Sigma-Aldrich, Saint Quentin Fallavier, France) and under centrifugation for 1 hour at 1,900 rpm and 37 °C. Following transduction, luciferase expression was determined by use of the Bright-Glo luciferase assay (Promega, Charbonnières-les-Bains, France) and a Centro LB 960 luminometer (Berthold Technologies). The assay was performed in triplicate.
For the kinetics, HCT116 cells were seeded in six-well plate and incubated 24 hours at 37 °C in 5% CO2. Transduction by ILV, IDLV, or MS2RLP was carried out in presence of 4 µg/ml Polybrene with 2 × 105 physical particles/cell. The transduction-supernatant is removed 5 hours later. From 4–24 hours post-transduction, cells were harvested and luciferase expression was analyzed using the One-Glo Luciferase assay (Promega) as above. The assay was performed in triplicate.
For the kinetics, HCT116 cells were seeded in six-well plate and incubated 24 hours at 37 °C in 5% CO2. Transduction by ILV, IDLV, or MS2RLP was carried out in presence of 4 µg/ml Polybrene with 2 × 105 physical particles/cell. The transduction-supernatant is removed 5 hours later. From 4–24 hours post-transduction, cells were harvested and luciferase expression was analyzed using the One-Glo Luciferase assay (Promega) as above. The assay was performed in triplicate.
10
Bioluminescent Coelenterazine Kinetics
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T4 polynucleotide kinase (3’-phosphatase minus, M0236) was obtained from New England Biolabs (NEB). 3’-phosphoadenosine 5’-phosphate and adenosine 3’-monophosphate were from Sigma-Aldrich. 100 μl reactions containing 100 mM Bis-tris propane pH 7.0, 1 mM DTT, 1mM EDTA, 200 nM coelenterazine sulfate, 10 nM of RLuc, and 1–20 nmoles of Coel-ST were performed at 25°C. Each reaction was initiated with the addition of the PAP sample and was followed by light emission. Relative light units per second (RLU/s) were measured in a Centro LB 960 luminometer (Berthold) plate reader.
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