Modulus 2, by Promega - Product details

1

Quantifying Cellular Glucose Metabolism

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Glucose uptake was measured using Glucose Uptake-GloTM kit (no. J1341, Promega) following the manufacturer’s protocol. Briefly, the analogue of glucose, 2-deoxyglucose (2DG), was added to the media and taken up by cells. When transported into cells, 2DG is phosphorylated to 2-deoxyglucose 6-phosphate (2DG6P) and further metabolization stimulates luciferase reactions and luminescence was measured by the microplate reader Modulus TM II (Turner Biosystems, Sunnyvale, CA).
Glucose consumption and lactate production were measured from the collected media when cells were in a high confluency. Metabolites were measured at the Analyzer machine (ABL90 FLEX Analyzer, Radiometer) at the Blood Bank of Landspitali (Reykjavik, Iceland).

Morera E., Steinhäuser S.S., Budkova Z., Ingthorsson S., Kricker J., Krueger A., Traustadottir G.A, & Gudjonsson T. (2019). YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation. In Vitro Cellular & Developmental Biology. Animal, 55(10), 838-853.

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2

Caspase-3/7 Cleavage Assay for Apoptosis

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To quantify apoptosis, cleavage of caspase 3/7 was measured by a luciferase assay (ApoTox-GloTM Triplex Assay, Promega, Madison, WI). Apoptosis was induced by incubating cells with 10 μM camptothecin (CPT) for 24 h according to the manufacturer’s protocol. After cellular lysis, luciferase was measured with a microplate reader ModulusTM II (Turner Biosystems, Sunnyvale, CA).

Morera E., Steinhäuser S.S., Budkova Z., Ingthorsson S., Kricker J., Krueger A., Traustadottir G.A, & Gudjonsson T. (2019). YKL-40/CHI3L1 facilitates migration and invasion in HER2 overexpressing breast epithelial progenitor cells and generates a niche for capillary-like network formation. In Vitro Cellular & Developmental Biology. Animal, 55(10), 838-853.

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3

SARS-CoV-2 Variant ADCC and ADCP Assays

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ADCC bioassay effector cells (Jurkat-FcγRIIIa-V158-NFAT-Luc) and ADCP bioassay effector cells (Jurkat-FcγRIIa-H131-NFAT-Luc) were obtained from Vazyme (Nanjing, China) and cultured in RPMI 1640 medium with 10% FBS. All cells were cultured at 37°C with 5% CO2.
The ADCC or ADCP reporter assay was performed in accordance with the manufacturer's instructions. The assays were performed using 293T-spike cells that expressed SARS-CoV-2 S protein as target cells. To obtain target cells, plasmids expressing viral S protein, pCAGGS-prototype-S, pCAGGS-BA.1-S, pCAGGS-BA.1.1-S, pCAGGS-BA.2-S, pCAGGS-BA.2.12.1-S, pCAGGS-BA.2.75-S, and pCAGGS-BA.4/5-S, were transfected into 293 T cells for 24 h, respectively. For all reporter assays, target cells (1.25 × 10⁴ per well) were incubated with plasma samples at 1/200 dilution. Effector cells (7.5 × 10⁴ per well) were then added to each well. After incubation at 37 °C for 24 h, 75 μl of Bright-Lite Reagent (Vazyme) were added to the wells and incubated for 5–10 min, and the relative light unit (RLU) was detected with Modulus II (Promega, CA, USA). The fold of induction was calculated as follows: RLU (induced − background)/RLU (no serum control − background).

Guo L., Zhang Q., Zhong J., Chen L., Jiang W., Huang T., Li Y., Zhang Y., Xu L., Wang X., Xiao Y., Wang Y., Dong X., Dong T., Peng Y., Zhang B., Xie Y., Gao H., Shen Z., Ren L., Cheng T, & Wang J. (2023). Omicron BA.1 breakthrough infections in inactivated COVID-19 vaccine recipients induced distinct pattern of antibody and T cell responses to different Omicron sublineages. Emerging Microbes & Infections, 12(1), 2202263.

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4

SARS-CoV-2 Variant ADCC and ADCP Assays

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ADCC bioassay effector cells (Jurkat-FcγRIIIa-V158-NFAT-Luc) and ADCP bioassay effector cells (Jurkat-FcγRIIa-H131-NFAT-Luc) were obtained from Vazyme (Nanjing, China) and cultured in RPMI 1640 medium with 10% FBS. All cells were cultured at 37°C with 5% CO2.
The ADCC or ADCP reporter assay was performed in accordance with the manufacturer's instructions. The assays were performed using 293T-spike cells that expressed SARS-CoV-2 S protein as target cells. To obtain target cells, plasmids expressing viral S protein, pCAGGS-prototype-S, pCAGGS-BA.1-S, pCAGGS-BA.1.1-S, pCAGGS-BA.2-S, pCAGGS-BA.2.12.1-S, pCAGGS-BA.2.75-S, and pCAGGS-BA.4/5-S, were transfected into 293 T cells for 24 h, respectively. For all reporter assays, target cells (1.25 × 10⁴ per well) were incubated with plasma samples at 1/200 dilution. Effector cells (7.5 × 10⁴ per well) were then added to each well. After incubation at 37 °C for 24 h, 75 μl of Bright-Lite Reagent (Vazyme) were added to the wells and incubated for 5–10 min, and the relative light unit (RLU) was detected with Modulus II (Promega, CA, USA). The fold of induction was calculated as follows: RLU (induced − background)/RLU (no serum control − background).

Guo L., Zhang Q., Zhong J., Chen L., Jiang W., Huang T., Li Y., Zhang Y., Xu L., Wang X., Xiao Y., Wang Y., Dong X., Dong T., Peng Y., Zhang B., Xie Y., Gao H., Shen Z., Ren L., Cheng T, & Wang J. (2023). Omicron BA.1 breakthrough infections in inactivated COVID-19 vaccine recipients induced distinct pattern of antibody and T cell responses to different Omicron sublineages. Emerging Microbes & Infections, 12(1), 2202263.

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5

Validating miR-634 Regulation of PIK3R1

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Diana-MICROT (http://www.microrna.gr/microT-CDS), miRDB (http://mirdb.org), TargetMiner (http://www.isical.ac.in) and TargetScan (http://www.targetscan.org) were used to predict the miR-634 targets. The miR-634 target region of the PIK3R1-3′UTR was inserted into the pmiR-RB-Report vector and named pmiR-PIK3R1-WT (RiboBio, Guangzhou, China). As a control, the plasmid containing the mutant sequence of the miR-634 target region of the PIK3R1 3′UTR was also inserted into the pmiR-RB-Report vector and named pmiR-PIK3R1-MUT. HEK293 cells were transiently co-transfected with the 100 ng pmiR-PIK3R1-WT or pmiR-PIK3R1-MUT and 30 nM miR-634 mimic or its negative control using Lipofectamine 3000 (Invitrogen, CA, USA) in 24-well plates, respectively. Each experiment was repeated 3 times. 48 hours after transfection, luciferase activity was determined with Dual-Luciferase® Assay System according to manufacturer’s protocol (Promega, WI, USA). Luminescence was measured using Modulus™ II (Turner Biosystems, USA).

Cui X., Wang S., Cai H., Lin Y., Zheng X., Zhang B, & Xia C. (2016). Overexpression of microRNA-634 suppresses survival and matrix synthesis of human osteoarthritis chondrocytes by targeting PIK3R1. Scientific Reports, 6, 23117.

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6

Quantifying Viral Infectivity Using Luciferase Assay

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Virus-containing supernatants were pre-cleared by low-speed centrifugation (5 min, 1,500 rpm) and filtered through a 0.45-μm syringe filter. TZM-bl indicator cells (CD4⁺, CCR5⁺, CXCR4⁺) were plated in a 24-well plate (1 mL; 5 × 10⁴ cells/well) and infected with 150 μL of viral supernatant. Typically, infections were performed in triplicate. Cells were lysed in the wells 48 hr later with 200 μL of lysis buffer (25 mM Tris-HCl [pH 7.8], 8.0 mM MgCl₂, 1.0 mM DTT, 1.0% Triton X-100, 15% glycerol). Luciferase activity in the lysate was determined by combining 5 μL of each lysate with 20 μL of luciferase substrate (Steady-Glo; Promega, Madison, WI, USA). Light emission was measured using a Modulus II microplate reader (Turner Biosystems, Sunnyvale, CA, USA). Values were normalized by reverse-transcriptase activity.

Sukegawa S., Miyagi E., Bouamr F., Farkašová H, & Strebel K. (2018). Mannose Receptor 1 Restricts HIV Particle Release from Infected Macrophages. Cell reports, 22(3), 786-795.

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7

Quantifying Viral Infectivity Using Luciferase Assay

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Virus-containing supernatants were pre-cleared by low-speed centrifugation (5 min, 1,500 rpm) and filtered through a 0.45-μm syringe filter. TZM-bl indicator cells (CD4⁺, CCR5⁺, CXCR4⁺) were plated in a 24-well plate (1 mL; 5 × 10⁴ cells/well) and infected with 150 μL of viral supernatant. Typically, infections were performed in triplicate. Cells were lysed in the wells 48 hr later with 200 μL of lysis buffer (25 mM Tris-HCl [pH 7.8], 8.0 mM MgCl₂, 1.0 mM DTT, 1.0% Triton X-100, 15% glycerol). Luciferase activity in the lysate was determined by combining 5 μL of each lysate with 20 μL of luciferase substrate (Steady-Glo; Promega, Madison, WI, USA). Light emission was measured using a Modulus II microplate reader (Turner Biosystems, Sunnyvale, CA, USA). Values were normalized by reverse-transcriptase activity.

Sukegawa S., Miyagi E., Bouamr F., Farkašová H, & Strebel K. (2018). Mannose Receptor 1 Restricts HIV Particle Release from Infected Macrophages. Cell reports, 22(3), 786-795.

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8

Measuring Antioxidant Activity via ABTS Assay

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The ABTS^+• cation-radical bleaching assay was carried out according to Re et al. [67 (link)], with some modifications. The test was performed in a Modulus^® II microplate reader (Turner Biosystems Inc., Sunnyvale, CA, USA). ABTS (7 mM in acetate buffer) and PDS (2.45 mM) were mixed with sonication (30 min) and stored at 4 °C (24 h) in the absence of light. ABTS^+• (900 μL) was taken and diluted in acetate buffer until an absorbance of 0.71 ± 0.02 at λ = 750 nm was obtained. Each extract (2 mg) was dissolved in methanol (2 mL) and diluted in acetate buffer (20 mM, pH 4.5). The diluted extract (10 μL) and ABTS^+• solution (190 μL) were deposited in each well of the plate, and the absorbance was measured for 60 min.

Porras S.M., Saavedra R.A., Sierra L.J., González R.T., Martínez J.R, & Stashenko E.E. (2023). Chemical Characterization and Determination of the Antioxidant Properties of Phenolic Compounds in Three Scutellaria sp. Plants Grown in Colombia. Molecules, 28(8), 3474.

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9

Quantification of Oxidative Stress Markers

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Quantification of malondialdehyde (MDA) (ab118970, Abcam, Cambridge, UK) and nitrite/nitrate NO (Item No. 780001, Cayman Chemical, Ann Arbor, MI, USA) in tissue samples were obtained according to the manufacturer’s instructions. Optical density was quantified using a Modulus II microplate reader (Turner Biosystems, Sunnyvale, CA, USA).

Asong-Fontem N., Panisello-Rosello A., Lopez A., Imai K., Zal F., Delpy E., Rosello-Catafau J, & Adam R. (2021). A Novel Oxygen Carrier (M101) Attenuates Ischemia-Reperfusion Injuries during Static Cold Storage in Steatotic Livers. International Journal of Molecular Sciences, 22(16), 8542.

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Modulus 2

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9 protocols using modulus 2

Quantifying Cellular Glucose Metabolism

Caspase-3/7 Cleavage Assay for Apoptosis

SARS-CoV-2 Variant ADCC and ADCP Assays

SARS-CoV-2 Variant ADCC and ADCP Assays

Validating miR-634 Regulation of PIK3R1

Quantifying Viral Infectivity Using Luciferase Assay

Quantifying Viral Infectivity Using Luciferase Assay

Measuring Antioxidant Activity via ABTS Assay

Quantification of Oxidative Stress Markers

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