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Glomax multi plate reader

Manufactured by Promega
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The GloMax-Multi plate reader is a compact and versatile multi-mode microplate reader designed for a variety of applications in the laboratory. It offers accurate measurements of luminescence, fluorescence, and absorbance in 96-well and 384-well microplates. The instrument provides reliable performance and flexible configuration options to meet the needs of diverse research and testing requirements.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions) Dual luciferase reporter assay, by Promega (3 mentions) Dual luciferase reporter assay system, by Promega (3 mentions) Nap 10 sephadex g 25 column, by GE Healthcare (2 mentions) Dual glo luciferase assay system, by Promega (2 mentions) Dual light reporter system, by Thermo Fisher Scientific (2 mentions) Apo one homogenous caspase 3 7 assay, by Promega (2 mentions) Black walled 96 well plates, by BD (2 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions) One glo reagent, by Promega (1 mentions) Microsoft excel, by GraphPad (1 mentions) Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions) Peimax transfection reagent, by Polysciences (1 mentions) 4egi 1, by Merck Group (1 mentions) Stop glo reagent, by Promega (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Luciferase assay reagent 2, by Promega (1 mentions) Ros glo, by Promega (1 mentions) L 012, by Fujifilm (1 mentions) Celltiter glo assay kit, by Promega (1 mentions)

27 protocols using glomax multi plate reader

1

RARE Assay in 96-well Format

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To run the RARE assay in a 96-well plate format, C3RL4 cells were seeded in complete BME in a white cell culture plate (Corning, NY) at 14,000 cells/well and grown overnight at 37°C and 5% CO2 . In the agonist-mode assay, compound-containing medium was added to each assay well to reach final compound concentrations ranging from 8 μM to 0.5 nM (1:4 series dilution, 8 concentrations). In the antagonist-mode assay, compound-containing medium was added to each assay well immediately followed by addition of retinol-containing medium to reach final compound concentrations ranging from 50 μM to 3 nM (1:4 series dilution, 8 concentrations) and 1 μM retinol, respectively. The incubation was continued, protected from light exposure, for 6 hr at 37°C and 5% CO2. To terminate cell culture and measure luciferase activity, One-Glo reagent (Promega, Madison, WI) was added (1:1 volume) directly to each well and the plates were incubated (protected from light exposure) at room temperature for 30 min. Luminescence readout for each well was measured on a GloMax Multi+ plate reader (Promega) and the Relative Luminescence Unit (RLU) was analyzed using the Instinct (Promega), Microsoft Excel and Prism (Graphpad, La Jolla, CA) software packages.
Chen Y., Sakamuru S., Huang R., Reese D.H, & Xia M. (2016). Identification of compounds that modulate retinol signaling using a cell-based qHTS assay. Toxicology in vitro : an international journal published in association with BIBRA, 32, 287-296.
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2

Calcein Leakage Assay for Lipid Vesicles

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Purified calcein-loaded LUVs were mixed with D-dfTAT in presence or absence of UNC7938 (1–100 μM) at a 1:50 peptide: lipid ratio for 1 h at room temperature in 100 mM NaCl, 10 mM NaH2PO4 pH5.5. UNC7938 stock solution Samples were centrifuged for 1 min at 4,000 rpm. To measure the amount of leaked calcein and separate soluble liposomes from released calcein, the supernatants were purified using an illustra NAP-10 Sephadex G-25 column (GE Healthcare) (the elution volumes of liposomes and free calcein were determined independently with pure samples). Fractions were collected in a 96-well plate and the fluorescence of calcein was measured using a Promega GloMax-Multi plate reader (Ex 490nm, Em 520–560nm). Note, since UNC7938 stock is dissolved in 100% DMSO. The control of D-dfTAT+ 0.1% DMSO (concentration of DMSO in the UNC7938 incubations) with liposomes was performed as a control. 100% leakage was established by treating liposomes with the detergent Triton X-100.
Allen J., Najjar K., Erazo-Oliveras A., Kondow-McConaghy H.M., Brock D.J., Graham K., Hager E.C., Marschall A.L., Dübel S., Juliano R.L, & Pellois J.P. (2019). Cytosolic delivery of macromolecules in live human cells using the combined endosomal escape activities of a small molecule and cell penetrating peptides. ACS chemical biology, 14(12), 2641-2651.
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3

Luciferase Assays for CHIKV Split Replication

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Luciferase assays for transfections with the CHIKVRep split replication system, CHILuc and LucCHI were performed as previously described [13 (link),26 (link)] with the Dual-Luciferase Reporter Assay (Promega) on a GloMax multi+ plate reader (Promega) (S4 and S5 Figs). The assays were carried out according to manufacturer’s instructions.
Tng P.Y., Carabajal Paladino L.Z., Anderson M.A., Adelman Z.N., Fragkoudis R., Noad R, & Alphey L. (2022). Intron-derived small RNAs for silencing viral RNAs in mosquito cells. PLoS Neglected Tropical Diseases, 16(6), e0010548.
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4

Measurement of NF-κB Activity in Caco-2 Cells

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An NF-κB luciferase reporter plasmid (500 ng; Ehrentraut et al., 2013 (link)) and a Renilla reporter plasmid (1 ng) were transfected into subconfluent Caco-2 cells using Lipofectamine LTX Reagent (Thermo Scientific). Luciferase activity was determined at 16 h using Promega dual luciferase reagents (Promega, Madison, WI), and luminescence was determined using the GloMax-Multi plate reader (Promega). Each experiment was performed in triplicate.
Ehrentraut S.F., Curtis V.F., Wang R.X., Saeedi B.J., Ehrentraut H., Onyiah J.C., Kelly C.J., Campbell E.L., Glover L.E., Kominsky D.J, & Colgan S.P. (2016). Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation. Molecular Biology of the Cell, 27(23), 3687-3694.
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5

Quantifying TLR2 Activation by BLPs

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TLR2 stimulation was assessed by reporter gene assay using human TLR2-expressing HEK293T cells (kindly provided by Dr. Tsuyoshi Sugiyama). HEK293T cells were transfected with a human TLR2-expressing plasmid, pNF-κB-TA-luc (a NF-κB-luciferase reporter plasmid), and pGL4.73 (constitutive Renila luciferase expression plasmid, internal control) using PEImax transfection reagent (Polysciences). After incubation for 48 h, cells were treated with various concentrations of BLPs or 100 ng/ml of Pam3CSK4 (a synthetic TLR2 ligand) for an additional 24 h. Luciferase activities were measured using the Dual-Glo luciferase assay system (Promega) and GloMax multiplate reader (Promega), according to the manufacturer’s instructions.
Sudo H., Tokunoh N., Tsujii A., Kawashima S., Hayakawa Y., Fukushima H., Takahashi K., Koshizuka T, & Inoue N. (2023). The adjuvant effect of bacterium-like particles depends on the route of administration. Frontiers in Immunology, 14, 1082273.
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6

Dual Luciferase Reporter Assay in CaOV3 Cells

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Twenty-four hours before transfection, CaOV3 cells were seeded in antibiotic-free media in 12-well plates, at a density of 30 000 cells/well. One hundred nanogram of reporter was transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Twenty-four hours after transfection, luciferase activity was assayed using the Dual Luciferase Reporter Assay (Promega) and a Glomax-Multi+ Plate Reader (Promega), following the manufacturer’s instructions. After subtracting background measurements, Renilla luciferase activity intensity (IRluc) was normalized over firefly luciferase activity (IFluc). The fold change expression of the reporters relative to psiCHECK2 was calculated as: (IRluc Reporter/IFluc Reporter)/(IRluc psiCHECK2/IFluc psiCHECK2). The assay was repeated least three times for each reporter and the P-value was calculated by Student’s t-test.
Chen X., Paranjape T., Stahlhut C., McVeigh T., Keane F., Nallur S., Miller N., Kerin M., Deng Y., Yao X., Zhao H., Weidhaas J, & Slack F. (2014). Targeted resequencing of the microRNAome and 3′UTRome reveals functional germline DNA variants with altered prevalence in epithelial ovarian cancer. Oncogene, 34(16), 2125-2137.
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7

Lipid Binding Affinity of Peptides

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To determine the difference in the binding affinity of the peptides to LUVs of different lipid compositions, liposome binding assays were conducted. The LUVs were incubated for 1 h with either peptide at the same peptide to lipid ratio as the leakage assay (1:50) using the buffer composition of 100 mM NaCl, 10 mM NaH2PO4 pH 5.5. Samples were centrifuged at 13,000 rpm for 3 min. The supernatant was removed to measure the amount of unbound peptide (using the fluorescence emission of TMR). To insure that quenching due to the proximity of the TMR fluorophore in the dfTAT constructs would not affect our results, the supernatant was reduced with TCEP (50 mM). The TMR fluorescence was measured using the red channel (Ex= 525nm, Em=580–640 nm) of a Promega GloMax-Multi plate reader (Promega). The amount of peptide bound to the MLVs was determined according to the following equation:
Pb=Ptot-P
Where Ptotal is the amount of peptide from the supernatant in the absence of MLVs, P is the fraction of unbound peptide at a particular lipid concentration and Pb is the fraction of bound peptide at a particular lipid concentration.
Najjar K., Erazo-Oliveras A., Mosior J.W., Whitlock M.J., Rostane I., Cinclair J.M, & Pellois J.P. (2017). Unlocking endosomal entrapment with supercharged arginine-rich peptides. Bioconjugate chemistry, 28(12), 2932-2941.
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8

Investigating the Mechanism of 4EGI-1 in Modulating Protein Translation

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Mechanistically, 4EGI-1 binds with eIF4E to specifically disrupt association of eIF4G, a large scaffold protein that recruits the 40S ribosomal subunit, while promoting and stabilizing the binding of 4E-BP1 (Sekiyama et al., 2015 (link)). 10T1/2 cells were seeded at a density of 4.0×105 cells/well in a 96-well plate and incubated at 37°C overnight. The next day, cells were treated with 50 μM 4EGI-1 (Sigma, 324517) or DMSO in DMEM supplemented with 10% FBS and 2 mM L-glutamine and incubated for 4 h at 37°C. Following treatment, cells were co-transfected with capped and poly(A)-tailed FLuc (100 ng) and RLuc reporter RNAs (10 ng) using Lipofectamine 2000 (Thermo Fisher, 11668–019). Cells were then re-treated with 50 μM 4EGI-1 or DMSO and incubated for a further 4 h at 37°C. After a total of 8 h of treatment (4 h prior to transfection and 4 h after transfection), cells were harvested in 1× Passive Lysis Buffer and luciferase activities were measured using a Dual-Luciferase Reporter Assay System as per manufacturer’s instructions (Promega, E1980) on a GloMax-Multi plate reader (Promega).
Tiu G.C., Kerr C.H., Forester C.M., Krishnarao P.S., Rosenblatt H.D., Raj N., Lantz T.C., Zhulyn O., Bowen M.E., Shokat L., Attardi L.D., Ruggero D, & Barna M. (2021). A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies. Developmental cell, 56(14), 2089-2102.e11.
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9

Measuring Cell Luciferase Activity

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Luciferase assays were performed as previously described60 (link) with the Dual-Luciferase Reporter Assay (Promega) on a GloMax multi+ plate reader (Promega). Briefly, cells were lysed with 30 µl 1× passive lysis buffer (Promega) each after two washes with ion-free phosphate buffered saline (1× PBS) and subjected to one freeze–thaw cycle at −80 °C. Luciferase assay reagent II (Promega) and stop & glo reagent (Promega) were prepared according to the manufacturer’s instructions and the luciferase activities were measured.
Tng P.Y., Carabajal Paladino L., Verkuijl S.A., Purcell J., Merits A., Leftwich P.T., Fragkoudis R., Noad R, & Alphey L. (2020). Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression. Communications Biology, 3, 413.
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10

Measuring Intracellular Reactive Oxygen Species

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PASMC were seeded into 96-well plates, grown to 80–90% confluence and serum-starved for 24hrs. Cell were then incubated with the luminol-derived superoxide probe 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2 H,3 H)-dione (L-012, 50 µM, Wako Pure Chemical Industries) [27] (link), in PBS at 37 °C. We also used an alternative chemi-luminescence ROS probe (ROS-Glo™, Promega) [28] to measure H2O2 production in IPA. Changes in luminescence induced by acute drug treatments were detected using a Promega GloMax Multi+ plate reader. Background measurements using ROS probe plus drug, but in the absence of cells/tissue, were subtracted from all equivalent test readings in the presence of cells/tissue. Each final value for each treatment for each batch of cells was the average of measurements from at least 8 identically treated wells in each plate.
MacKay C.E., Shaifta Y., Snetkov V.V., Francois A.A., Ward J.P, & Knock G.A. (2017). ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1. Free Radical Biology & Medicine, 110, 316-331.
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