Rluc-ATP4A was expressed in vitro using the TnT SP6 Quick Coupled Transcription/Translation kit (Promega), based on transcription by the SP6 phage RNA polymerase and translation by a rabbit reticulocyte lysate cell-free expression system. Nluc-ATP4B was expressed in eukaryotic cells, using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA). In the Expi293 expression system, recombinant protein expression is achieved by high efficiency transfection of Expi293F, a derivative of HEK293 cells, adapted to growth in suspension in a defined composition, serum free medium. After 48 h of growth with agitation, transfected Expi293F cells were pelleted and lysed with passive lysis buffer (Thermo Fisher Scientific). Expression of recombinant antigens was assessed by quantification of luciferase activity in the lysates after the addition of Renilla luciferase assay system substrate or NanoGlow substrate (Promega), reconstituted according to the manufacturer instructions, for ATP4A and ATP4B, as appropriate. Luciferase activity was measured using a Berthold Centro xS960 luminometer (Berthold, Germany) and expressed in light units (LU) emitted over a time interval of 2 s. Recombinant antigen preparations were aliquoted and stored frozen at −80 °C.
Centro xs960 luminometer
Manufactured by Berthold Technologies
Sourced in Germany
The Centro xS960 luminometer is a compact and versatile instrument designed for luminescence-based assays. It features a high-sensitivity detector, programmable parameters, and intuitive software for data analysis. The core function of the Centro xS960 is to accurately measure luminescent signals, which is a fundamental technique in various fields of research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Multidrop dispenser, by Thermo Fisher Scientific (4 mentions)
Enduren, by Promega (4 mentions)
Bright glo luciferase assay system, by Promega (3 mentions)
One glo luciferase assay system, by Promega (2 mentions)
Passive lysis buffer (plb), by Thermo Fisher Scientific (1 mentions)
Renilla luciferase assay system substrate, by Promega (1 mentions)
Nanoglow substrate, by Promega (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
8 protocols using centro xs960 luminometer
1
In Vitro and Eukaryotic Expression of ATP4A and ATP4B
2
SARS-CoV-2 S Protein Cell-Cell Fusion Assay
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The DSP assay was performed as described previously (24 (link)). Briefly, effector cells expressing S protein and target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10-cm plates and incubated overnight (Fig. S1 a and b). Cells were treated with 6 μM EnduRen (Promega), a substrate for Renilla luciferase (RL), for 2 h. To test the effect of inhibitor, 0.25 μL of compound library or 1 μL of selected inhibitor dissolved in DMSO was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single-cell suspension (effector and target cells) was added to the 384-well plates by using a Multidrop dispenser (Thermo Fisher Scientific). After incubation at 37°C in 5% CO2 for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Bad Wildbad, Germany).
3
SARS-CoV-2 Entry Inhibition Assay
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For the DSP assay using 293FT cells, effector cells expressing S protein with DSP8-11 and target cells expressing CD26 or ACE2, and TMPRSS2 with DSP1-7 were seeded in 12-well cell culture plates (2 × 105 cells/500 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 μL of each single cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Scientific, Waltham, MA, USA). After incubation at 37 °C for 4 h, the RL activity was measured using a Centro xS960 luminometer (Berthold, Germany). For the DSP assay using Calu-3 or H3255 cells, target cells were seeded in 384-well plates (2 × 104 cells/50 μL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 μM EnduRen. One microliter of each protease inhibitor or anticoagulant dissolved in DMSO was added to the 384-well plates with 9 μL of culture medium. Next, 40 μL of single cell suspension (effector cells) was added to the wells using a Multidrop dispenser.
4
MERS-CoV Membrane Fusion Quantification
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MERS-CoV-S-mediated membrane fusion was quantitatively evaluated via DSP assay using 293FT cells as previously described (Yamamoto et al., 2016 (link)). The dimerization of DSP1-7 and DSP8-11 after cell-cell fusion can be quantified based on the values of fluorescence/luminescence upon formation of tight DSP complexes. The effector cells express MERS-CoV-S protein with DSP8-11, while the target cells express the MERS-CoV receptor and transmembrane serine protease 2 (TMPRSS2) with DSP1-7. The cells were grown in 10 cm culture dishes (4 × 106 cells/10 ml) 24 h before the assays. Cells were treated with 6 µM EnduRen (Promega, Madison, WI, United States), a substrate for Renilla luciferase, for 2 h to activate EnduRen. Each peptide was dissolved in dimethyl sulfoxide (DMSO) and added to 384-well plates (Greiner Bioscience, Frickenhausen, Germany), then 50 µl of each single-cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Fisher Scientific). After incubation at 37°C for 4 h, luciferase activity was measured using a Centro xS960 luminometer (Berthold, Germany).
5
SARS-CoV-2 Pseudovirus Infection Assay
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293T cells were transfected with an expression plasmid for SARS-CoV-2-S, VSV-G, or control expression plasmid by calcium-phosphate precipitation. At 16 h posttransfection, the cells were inoculated with a replication-deficient VSV, VSV-ΔG-Luciferase, which lacks the VSV-G gene and encodes firefly luciferase, at an MOI=1 as described previously (Tani et al., 2010 (link)). After 2 h of incubation, cells were washed with DMEM and further incubated for 16 h before supernatants containing the pseudotyped viral particles were harvested. Cellular debris was removed from the supernatants using a syringe filter with a 0.2 µm size pore (Millipore, Bedford, MA, USA).
For an infection assay, target Calu-3 cells were seeded in 96-well plates (5×104 cells/100 µL) one day before the assay. Cells were pre-treated with peptides for 1 h before infection. Pseudotyped viral particles were added to cells with the peptides. After 2 h of incubation, the culture supernatant was removed, and cells were washed with EMEM. Cells were further incubated in EMEM containing 10% FBS without peptides and pseudotyped viral particles. At 16 h postinfection, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega) and Centro xS960 luminometer (Berthold).
For an infection assay, target Calu-3 cells were seeded in 96-well plates (5×104 cells/100 µL) one day before the assay. Cells were pre-treated with peptides for 1 h before infection. Pseudotyped viral particles were added to cells with the peptides. After 2 h of incubation, the culture supernatant was removed, and cells were washed with EMEM. Cells were further incubated in EMEM containing 10% FBS without peptides and pseudotyped viral particles. At 16 h postinfection, luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega) and Centro xS960 luminometer (Berthold).
6
Replication-deficient VSV Production and Infection
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To produce replication-deficient vesicular stomatitis virus (VSV), BHK cells expressing T7 RNA polymerase were transfected with T7 promoter-driven expression plasmids for VSV proteins (pBS-N/pBS-P/pBS-L/pBS-G) and pΔG-Luci (a plasmid encoding VSV genomic RNA lacking the G gene and encoding firefly luciferase) as described previously [32 (link),33 (link)]. At 48 h post-transfection, the supernatants were harvested. The 293T cells were then transfected with an expression plasmid for S protein or VSV G using calcium phosphate precipitation. At 16 h post-transfection, cells were infected with replication-deficient VSV at a multiplicity of infection (MOI) of 1. At 2 h post-infection, cells were washed and incubated for another 16 h before supernatants containing pseudovirus were harvested. For the infection assay, VeroE6-TMPRSS2 cells were seeded in 96-well plates (2 × 104 cells/well) and incubated overnight. Cells were pretreated with inhibitors for 1 h prior to pseudovirus infection. Luciferase activity was measured 16 h after infection using the Bright-Glo Luciferase Assay System or ONE-Glo Luciferase Assay System (Promega) and the Centro xS960 luminometer (Berthold, Bad Wildbad, Germany).
7
SARS-CoV-2 Membrane Fusion Assay
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The DSP assay using 293FT cells was performed as described previously (Yamamoto et al., 2020 (link)) to monitor SARS-CoV-2-S-mediated membrane fusion. Briefly, effector cells expressing SARS-CoV-2-S protein with DSP8-11, target cells expressing ACE2, and transmembrane serine protease 2 (TMPRSS2) with DSP1-7 were seeded in 10 cm culture dishes (4×106 cells/10 mL) one day before the assay. Two hours before the DSP assay, cells were treated with 6 µM EnduRen (Promega, Madison, WI, USA), a substrate for Renilla luciferase, to activate EnduRen. One microliter of each peptide dissolved in dimethyl sulfoxide (DMSO) was added to the 384-well plates (Greiner Bioscience, Frickenhausen, Germany). Next, 50 µL of each single-cell suspension (effector and target cells) was added to the wells using a Multidrop dispenser (Thermo Fisher Scientific, Waltham, MA, USA). After incubation at 37°C for 4 h, luciferase activity was measured using a Centro xS960 luminometer (Berthold, Bad-Wildbad, Germany).
8
Production and Infection of Replication-Deficient VSV
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For producing a replication-deficient VSV, BHK cells expressing T7 RNA polymerase were transfected with T7 promoter-driven expression plasmids for VSV proteins (pBS-N/pBS-P/pBS-L/pBS-G) and pΔG-Luci (a plasmid encoding VSV genomic RNA which lacks the G gene and encodes firefly luciferase) as described previously (60 (link), 61 (link)). At 48 h posttransfection, the supernatants were harvested. 293T cells were then transfected with an expression plasmid for S or VSV G by using calcium phosphate precipitation. At 16 h posttransfection, the cells were inoculated with a replication-deficient VSV at a multiplicity of infection (MOI) of 1. At 2 h postinfection, the cells were washed and further incubated for 16 h before the supernatant containing the pseudovirus was harvested. For the infection assay, cells were seeded in 96-well plates (2 × 104 cells/well) and incubated overnight. The cells were pretreated with inhibitors for 1 h before the pseudovirus infection. Luciferase activity was measured at 16 h postinfection by using the Bright-Glo luciferase assay system or ONE-Glo luciferase assay system (Promega) and a Centro xS960 luminometer (Berthold).
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