Lumistar omega microplate luminometer

Manufactured by BMG Labtech

Sourced in Germany

The LUMIstar Omega is a microplate luminometer manufactured by BMG LABTECH. It is designed to measure luminescence-based assays in microplates. The instrument provides high-performance luminescence detection capabilities, with a wide linear dynamic range and rapid measurement speeds.

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Lab products found in correlation

Gsh gssg glo assay kit, by Promega (2 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions) Caspase 9, by Cell Signaling Technology (2 mentions) Caspase glo 3 7 assay system, by Promega (2 mentions) Bright glo luciferase reagent, by Promega (2 mentions) D luciferin, by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Celltiter blue reagent, by Promega (1 mentions) Fluostar omega microplate spectrophotometer, by BMG Labtech (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Poly adenosine diphosphate ribose polymerase parp, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Tc20 automatic cell counter, by Bio-Rad (1 mentions) Antibodies to β actin, by Santa Cruz Biotechnology (1 mentions)

8 protocols using lumistar omega microplate luminometer

Cytotoxicity Assay of CAR-T Cells

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Luciferase-expressing HeLa or HEK293 cells were seeded into a U-bottomed 96-well plate with D-luciferin (75 μg/mL; Sigma-Aldrich) for cytotoxicity assessment. After that, CAR-expressing T cells were added to the target cells at 20:1, 10:1, 5:1, and 2.5:1 effector: target ratios and incubated at 37 °C in 5% CO₂ for 2 h. The LUMIstar Omega microplate luminometer (BMG Labtech; Ortenberg, Germany) was used to measure luminescence as relative light units (RLUs). In each assay, for baseline lysis, one group of target cells was cultured alone, and for maximum lysis, another group was cultured in 1% Triton X-100 (Sigma-Aldrich). The percentage of the specific lysis was calculated according to the following formula: % specific lysis = 100 × (spontaneous cell death RLU − sample RLU)/(spontaneous death RLU − maximal killing RLU).

Yekehfallah V., Pahlavanneshan S., Sayadmanesh A., Momtahan Z., Ma B, & Basiri M. (2022). Generation and Functional Characterization of PLAP CAR-T Cells against Cervical Cancer Cells. Biomolecules, 12(9), 1296.

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Measuring Intracellular Glutathione Redox

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Relative changes in intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined with GSH/GSSG-Glo Assay kit (Promega, WI). Briefly, live cells collected from duplicate bioreactors were rinsed and resuspended in HBSS buffer, then seeded at 10,000 cells/well in a 96-well luminometer-compatible plate. 25 µl of either total glutathione lysis reagent or oxidized glutathione lysis reagent was added to the wells containing cells and incubated at room temperature on a plate shaker for 5 min. Then 50 µl of freshly prepared luciferin generation reagent was added to all the wells followed by 30 min incubation at room temperature. 100 µl of luciferin detection reagent was then added to each well. Luminescence was measured after 15 min of incubation using a LUMIstar Omega Microplate Luminometer (BMG Labtech). No cell and HBSS buffer only wells were used for background luminescence detection. Relative Luminescence Unit (RLU) for GSH levels for each bioreactor samples were determined by subtracting RLU of GSSG from RLU of total glutathione.

Sinharoy P., Aziz A.H., Majewska N.I., Ahuja S, & Handlogten M.W. (2020). Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells. Scientific Reports, 10, 16620.

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Inflammasome-Activated Caspase-1 Assay

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For each sample, an equal volume of supernatant was transferred into 96-well plates and equilibrated at room temperature for 5 min. To detect inflammasome-related caspase-1 activity, an equal volume of the Caspase-Glo Inflammasome Reagent (Promega, Madison, WI, USA) was added in each well according to the manufacturer’s instructions. The plate was incubated at room temperature for 1 h in the dark and the relative luminescence units (RLU) of assays were determined in the LUMIstar Omega microplate luminometer (BMG Labtech, Ortenberg, Germany) according to the manufacturer’s directions.
For cell viability, cells were incubated with Cell Titer Blue Reagent (Promega, Madison, WI, USA) at 37 °C for 1 h according to the manufacturer’s instruction. The relative fluorescence (560Excitation/590Emission) was detected with the FLUOstar Omega microplate spectrophotometer (BMG Labtech, Ortenberg, Germany) following the manufacturer’s instructions.

Scalavino V., Piccinno E., Valentini A.M., Schena N., Armentano R., Giannelli G, & Serino G. (2023). miR-369-3p Modulates Intestinal Inflammatory Response via BRCC3/NLRP3 Inflammasome Axis. Cells, 12(17), 2184.

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Apoptosis Induction in Lymphoma Cells

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Cell death rates were determined by counting viable cells in trypan blue‐stained samples on a TC20 automatic cell counter (Bio‐Rad Laboratories). caspase 3/7 enzymatic activities were measured in cells treated with increasing concentrations (16‐64 μM) of CIS or DZ‐CIS for 12 hours by the Caspase‐Glo 3/7 Assay System (Promega, Madison, Wisconsin). Luminescence intensity was measured by LUMIstar Omega microplate luminometer (BMG Labtech, BioTek, Winooski, Vermont). Western blot analysis of caspase activation in whole cell lysates from treated lymphoma cells used antibodies to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), caspase 9, caspase 3, and poly(adenosine diphosphate‐ribose) polymerase (PARP) from Cell Signaling Technology (Danvers, Massachusetts) and horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, Texas).

Mrdenovic S., Zhang Y., Wang R., Yin L., Chu G.C., Yin L., Lewis M., Heffer M., Zhau H.E, & Chung L.W. (2019). Targeting Burkitt lymphoma with a tumor cell–specific heptamethine carbocyanine‐cisplatin conjugate. Cancer, 125(13), 2222-2232.

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Quantifying Intracellular Glutathione Redox Status

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Relative changes in intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined with the GSH/GSSG-Glo Assay kit (Promega, WI, USA). Briefly, the cells were collected and resuspended in HBSS buffer, then plated in a 96-well luminometer-compatible plate at 10,000 cells/well. 25 μl of either total GSH lysis reagent or GSSG reagent was added to the wells and incubated at room temperature on a shaker for 5 min. Then 50 μl of freshly prepared luciferin generation reagent was added to each well followed by 30 min incubation at room temperature. 100 μl of luciferin detection reagent was then added to each well. After 15 min of incubation, luminescence was measured using a LUMIstar Omega Microplate Luminometer (BMG Labtech). Wells without cell and only HBSS buffer were used for background luminescence detection. Relative luminescence unit (RLU) for GSH levels was determined by subtracting RLU of GSSG from RLU of total glutathione. The ratio GSH/GSSG was calculated using the formula as follows.

\begin{matrix} Ratio GSH / GSSG = \frac{μ M total - (μ M GSSG \times 2)}{μ M GSSG} . \end{matrix}

Wang R., Luo D., Li Z, & Han H. (2021). Dimethyl Fumarate Ameliorates Nucleus Pulposus Cell Dysfunction through Activating the Nrf2/HO-1 Pathway in Intervertebral Disc Degeneration. Computational and Mathematical Methods in Medicine, 2021, 6021763.

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Caspase-3/7 Activity Assay and Western Blot

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For the caspase activity assay, cells treated with DZ-CIS for 24 h were measured for caspase 3/7 enzymatic activities by the Caspase-Glo® 3/7 Assay System (Promega, Madison, WI) with the recommended protocol by the manufacturer. Luminescence intensity was acquired using a LUMIstar Omega microplate luminometer (BMG Labtech, BioTek, Winooski, VT). For western blot analysis, our previously reported protocol was used [24 ]. Antibodies to poly ADP-ribose polymerase (PARP), caspase 3, and caspase 9 were from Cell Signaling Technology (Danvers, MA). Antibodies to β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were used.

Mrdenovic S., Wang Y., Yin L., Chu G.C., Ou Y., Lewis M.S., Heffer M., Posadas E.M., Zhau H.E., Chung L.W., Edderkaoui M., Pandol S.J., Wang R, & Zhang Y. (2023). A cisplatin conjugate with tumor cell specificity exhibits antitumor effects in renal cancer models. BMC Cancer, 23, 499.

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Pseudovirion Infection Assay in SupT1 Cells

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Viruses were propagated in 293T cells reverse-transfected with pLAIΔenvLuc2Δvif, A3H plasmid, and L-VSV-G for pseudotyping. Virus-containing supernatants were harvested 48 h after transfection, transferred to a V-bottom plate, and clarified of cells and debris by centrifugation at 1000× g for 3 min at 25 °C. An amount of 10 µL of supernatant was added to flat-bottom 96-well plates containing SupT1 cells (3.75 × 10⁴ and 90 µL/well) pretreated with 20 µg/mL DEAE/Dextran and mixed by repipetting. An amount of 5 µL of supernatant was saved for quantification of reverse transcriptase (RT) activity, as described previously [29 (link)]. Infected SupT1 cells were incubated for 48 h and lysed in 100 µL of Bright-Glo Luciferase Reagent (Promega, E2610, Madison, WI, USA). Infection was assessed by luciferase activity using a LUMIstar Omega microplate luminometer (BMG Labtech, Ortenberg, Germany), and raw luciferase values were normalized to 2000 mU RT activity.

Chesarino N.M, & Emerman M. (2020). Polymorphisms in Human APOBEC3H Differentially Regulate Ubiquitination and Antiviral Activity. Viruses, 12(4), 378.

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Lentiviral Infectivity Assay Using APOBEC3

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Viruses were propagated in HEK293T cells by reverse transfection of the relevant pLAIΔenvLuc2Δvif and APOBEC3 plasmids, and L-VSV-G for pseudotyping (Bartz and Vodicka, 1997 (link)). Transfected cells were incubated for 48 hours until the viral supernatant was harvested and clarified of cellular debris by centrifugation in a V-bottom plate (1,000×G for 3 min at room temperature). 5 μL of viral supernatant was saved for quantitation of reverse transcriptase (RT) activity for normalization, as previously described (Vermeire et al., 2012 ). A standard 10 μL of viral supernatant was directly transferred to 96-well plates containing 90 μL of SupT1 cells (3.75×10⁴ cells/well) pretreated with 20 μg/mL DEAE-Dextran. SupT1 cells were incubated for 48 hours at 37 °C and 5% CO₂ prior to lysis with 100 μL of Bright-Glo Luciferase Reagent (Promega #E2610). Lysate was then assessed for luciferase activity using a LUMIstar Omega microplate luminometer (BMG Labtech). Raw luciferase values were normalized to 2,000 mU RT activity. Percent infectivity values were calculated relative to no APOBEC3 vector control (where no APOBEC3 was set to 100%).

Binning J.M., Chesarino N.M., Emerman M, & Gross J.D. (2019). Structural basis for a species-specific determinant of an SIV Vif protein towards hominid APOBEC3G antagonism. Cell host & microbe, 26(6), 739-747.e4.

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