Centroxs luminometer

Manufactured by Berthold Technologies

The CentroXS luminometer is a compact and versatile instrument designed for measuring luminescence in a variety of applications. It utilizes a sensitive photomultiplier tube to detect and quantify light output from luminescent samples. The CentroXS provides accurate and reliable luminescence measurements, making it a useful tool for researchers and scientists working in fields such as biochemistry, cell biology, and molecular biology.

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Lab products found in correlation

Pei max 40000, by Polysciences (3 mentions) Renilla luciferase assay reagent, by Promega (2 mentions) Prism v9, by GraphPad (1 mentions) Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions) Complete protease inhibitor mixture, by Roche (1 mentions) Streptactin beads, by GE Healthcare (1 mentions) Renilla lysis buffer, by Promega (1 mentions) Streptactin sepharose high performance, by GE Healthcare (1 mentions) Nanoluc substrate, by Promega (1 mentions) Nano glo, by Promega (1 mentions) Cell culture microplates, by Greiner (1 mentions) Cell titer glo luminescent viability assay kit, by Promega (1 mentions) Bright glo luciferase substrate, by Promega (1 mentions)

7 protocols using centroxs luminometer

Cell Viability Assay of Knockout Cell Lines

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A total of 8.0 × 10⁵ cells of each KO cell line were seeded in a white 96-well plate and incubated at 37°C and 5% CO₂ for 24 h. Cell media was replaced with DMEM and incubated at 37 °C and 5% CO₂ for 72 h. Cell viability was measured using Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, G7750). Luminescence was measured on a Centro XS luminometer (Berthold; integration time, 0.5 s). Wild-type cells served as the reference and significance of cell viability was calculated against the mock KO using an ordinary one-way nonparametric ANOVA Kruskal–Wallis with Dunn’s multiple comparisons test using GraphPad Prism v9.

Kim D.K., Weller B., Lin C.W., Sheykhkarimli D., Knapp J.J., Dugied G., Zanzoni A., Pons C., Tofaute M.J., Maseko S.B., Spirohn K., Laval F., Lambourne L., Kishore N., Rayhan A., Sauer M., Young V., Halder H., la Rosa N.M., Pogoutse O., Strobel A., Schwehn P., Li R., Rothballer S.T., Altmann M., Cassonnet P., Coté A.G., Vergara L.E., Hazelwood I., Liu B.B., Nguyen M., Pandiarajan R., Dohai B., Coloma P.A., Poirson J., Giuliana P., Willems L., Taipale M., Jacob Y., Hao T., Hill D.E., Brun C., Twizere J.C., Krappmann D., Heinig M., Falter C., Aloy P., Demeret C., Vidal M., Calderwood M.A., Roth F.P, & Falter-Braun P. (2022). A proteome-scale map of the SARS-CoV-2–human contactome. Nature Biotechnology, 41(1), 140-149.

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Affinity Purification of Viral Proteins

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HEK-293T (1–2×10⁷) cells were infected with WSN or P908/WSN recombinant viruses at a m.o.i. of 3. Six hours post-infection, cells were lysed in 0.5 ml of lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM of KCl, 0.5% Igepal), supplemented with Complete Protease Inhibitor Mixture (Roche). Cell lysates were processed and incubated wih StrepTactin beads (StrepTactin Sepharose High Performance, GE Healthcare) as described in [51] . After three washes with 1 ml of lysis buffer, protein complexes were eluted from StrepTactin beads with desthiobiotin (IBA). Purification samples were either diluted in Laemmli sample buffer and analyzed by western-blot, or diluted in Renilla lysis buffer (Promega) and submitted to Gaussia princeps luciferase enzymatic activity measurement, using the Renilla luciferase assay reagent (Promega) and a Berthold Centro XS luminometer.

Fournier G., Chiang C., Munier S., Tomoiu A., Demeret C., Vidalain P.O., Jacob Y, & Naffakh N. (2014). Recruitment of RED-SMU1 Complex by Influenza A Virus RNA Polymerase to Control Viral mRNA Splicing. PLoS Pathogens, 10(6), e1004164.

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NanoLuc Protein Complementation Assay

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HEK293T cells were seeded at 6x10⁴ cells per well in 96-well, flat-bottom, cell culture microplates (Greiner Bio-One, #655083), and cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10% fetal calf serum at 37 °C and 5% CO₂. Twenty-four hour later, cells were transfected with 100 ng of each N2H plasmid (pDEST-N2H-N1, -N2, -C1 or -C2) using linear polyethylenimine (PEI) to co-express the protein pairs fused with complementary NanoLuc fragments, F1 and F2. The DNA/PEI ratio used for transfection was 1:3 (mass:mass). Twenty-four hour after transfection, the culture medium was removed and 50 μL of 100× diluted NanoLuc substrate (Promega, #N1110) was added to each well of a 96-well microplate containing the transfected cells. Plates were incubated for 3 min at room temperature. Luciferase enzymatic activity was measured using a TriStar or CentroXS luminometer (Berthold; 2 s integration time).
The stock solution of PEI HCl (PEI MAX 40000; Polysciences Inc; Cat# 24765) was prepared according to the manufacturer’s instructions. Briefly, 200 mg of PEI powder added to 170 mL of water, stirred until complete dissolution, and pH was adjusted to 7 with 1 M NaOH. Water was added to obtain a final concentration of 1 mg/mL, and the stock solution was filtered through a 0.22 µm membrane.

Choi S.G., Olivet J., Cassonnet P., Vidalain P.O., Luck K., Lambourne L., Spirohn K., Lemmens I., Dos Santos M., Demeret C., Jones L., Rangarajan S., Bian W., Coutant E.P., Janin Y.L., van der Werf S., Trepte P., Wanker E.E., De Las Rivas J., Tavernier J., Twizere J.C., Hao T., Hill D.E., Vidal M., Calderwood M.A, & Jacob Y. (2019). Maximizing binary interactome mapping with a minimal number of assays. Nature Communications, 10, 3907.

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Nanoluciferase-based Protein-Protein Interactions

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HEK293T cells were seeded at 6x10⁴ cells per well in 96-well, flat-bottom, cell culture microplates (Greiner Bio-One, #655083), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum at 37 °C and 5% CO₂. 24 h later, cells were transfected with 100 ng of each N2H plasmid (pDEST-N2H-N1, -N2, -C1 or -C2) using linear polyethylenimine (PEI) to co-express proteins fused with complementary NanoLuc fragments, F1 and F2. The stock solution of PEI HCl (PEI MAX 40000; Polysciences Inc; Cat# 24765) was prepared according to the manufacturer’s instructions. Briefly, 200 mg of PEI HCl powder were added to 170 mL of water, stirred until complete dissolution, and pH was adjusted to 7 with 1 M NaOH. Water was added to obtain a final concentration of 1 mg/mL, and the stock solution was filtered through a 0.22 μm membrane. The DNA/PEI ratio used for transfection was 1:3 (mass:mass). 24 h after transfection, the culture medium was removed and 50 μL of 100x diluted NanoLuc substrate (Furimazine, Promega Nano-Glo, N1120) was added to each well of a 96-well microplate containing the transfected cells. Plates were incubated for 3 min at room temperature. Luciferase enzymatic activity was measured using a TriStar or CentroXS luminometer (Berthold; 2 s integration time).

Trepte P., Secker C., Kostova S., Maseko S.B., Choi S.G., Blavier J., Minia I., Ramos E.S., Cassonnet P., Golusik S., Zenkner M., Beetz S., Liebich M.J., Scharek N., Schütz A., Sperling M., Lisurek M., Wang Y., Spirohn K., Hao T., Calderwood M.A., Hill D.E., Landthaler M., Olivet J., Twizere J.C., Vidal M, & Wanker E.E. (2023). AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor. bioRxiv.

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siRNA-mediated knockdown of ExoRDec genes in A549 cells to study influenza virus replication

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siRNAs were purchased from Dharmacon (ON-TARGETplus SMARTpools and Non-Target Control pool). Individual siRNAs targeting ERI1 were purchased from Sigma Aldrich. A549 cells were transfected with 25 nM siRNA with the Interferine transfection reagent (Polyplus). siRNAs targeting FANCG, COPS5 and NUP62 factors, known from the literature to be required for IAV replication, were used as controls (43 (link),44 (link)). At 48 h post-transfection, cells were infected with the A/WSN/33(H1N1) at a multiplicity of infection (moi) of 10⁻⁴ pfu/cell or with A549 cell-adapted A/Bretagne/7608/2009 (H1N1pdm09) and A/Centre/1003/2012(H3N2), or with A/Paris/650/2004(H1N1) (moi 10⁻³ pfu/cell) virus for 24 h. Plaque assays with MDCK-SIAT cells were performed as described in (45 (link)). Cell viability was determined using the CellTiter-Glo Luminescent Viability Assay kit according to the manufacter's instructions (Promega). To control the efficiency of siRNAs targeting ExoRDec genes, siRNA-treated A549 cells were transfected with plasmids encoding ExoRDec proteins fused with the full-length Gaussia luciferase (pGlucFL-ExoN) using linear PEI (polyethylenimine). The luciferase activity was measured 24h later in cell lysates using the Renilla luciferase assay reagent (Promega) and a Berthold CentroXS luminometer as described before for GPCA.

Declercq M., Biquand E., Karim M., Pietrosemoli N., Jacob Y., Demeret C., Barbezange C, & van der Werf S. (2020). Influenza A virus co-opts ERI1 exonuclease bound to histone mRNA to promote viral transcription. Nucleic Acids Research, 48(18), 10428-10440.

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Quantification of SARS-CoV-2 pseudovirus entry

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HEK-293T cells stably expressing hACE2 were transduced in suspension by mixing 50 µl of threefold serial dilutions of S-pseudotyped lentiviruses with 50 µl of cells at 4 × 10 5 cells per ml in 96-well white culture plates 65 . At 60-72 h post-transduction, 100 µl of Bright-Glo luciferase substrate (Promega) was added to the wells and luminescence was measured using a Berthold Centro XS luminometer.

Temmam S., Vongphayloth K., Baquero E., Munier S., Bonomi M., Regnault B., Douangboubpha B., Karami Y., Chrétien D., Sanamxay D., Xayaphet V., Paphaphanh P., Lacoste V., Somlor S., Lakeomany K., Phommavanh N., Pérot P., Dehan O., Amara F., Donati F., Bigot T., Nilges M., Rey F.A., van der Werf S., Brey P.T, & Eloit M. (2022). Bat coronaviruses related to SARS-CoV-2 and infectious for human cells. Nature, 604(7905).

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Nanoluc Luciferase Assay in HEK293T Cells

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HEK293T cells were seeded at 6x10 4 cells per well in 96-well, flat-bottom, cell culture microplates (Greiner Bio-One, #655083), and cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10% fetal calf serum at 37 °C and 5% CO2. 24 h later, cells were transfected with 100 ng of each N2H plasmid (pDEST-N2H-N1, -N2, -C1 or -C2) using linear polyethylenimine (PEI) to co-express proteins fused with complementary NanoLuc fragments, F1 and F2. The stock solution of PEI HCl (PEI MAX 40000; Polysciences Inc; Cat# 24765) was prepared according to the manufacturer's instructions. Briefly, 200 mg of PEI HCl powder were added to 170 mL of water, stirred until complete dissolution, and pH was adjusted to 7 with 1 M NaOH. Water was added to obtain a final concentration of 1 mg/mL, and the stock solution was filtered through a 0.22 µm membrane. The DNA/PEI ratio used for transfection was 1:3 (mass:mass). 24 h after transfection, the culture medium was removed and 50 µL of 100x diluted NanoLuc substrate (Furimazine, Promega Nano-Glo, N1120) was added to each well of a 96-well microplate containing the transfected cells. Plates were incubated for 3 min at room temperature. Luciferase enzymatic activity was measured using a TriStar or CentroXS luminometer (Berthold; 2 s integration time).

Trepte P., Secker C., Choi S.G., Olivet J., Ramos E.S., Cassonnet P., Golusik S., Zenkner M., Beetz S., Sperling M., Wang Y., Hao T., Spirohn K., Twizere J., Calderwood M.A., Hill D.E., Jacob Y., Vidal M., & Wanker E.E. (2021). A quantitative mapping approach to identify direct interactions within complexomes.

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