Nunc microwell 96 well optical bottom plates

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Nunc MicroWell 96-Well Optical-Bottom Plates are a type of microplate designed for use in optical-based assays. The plates feature a transparent optical bottom that allows for the detection and measurement of signals from the wells, enabling applications such as absorbance, fluorescence, and luminescence experiments.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Tcs sp8x inverted confocal microscope, by Leica (2 mentions) Ionomycin, by Merck Group (2 mentions) Phorbol myristate acetate (pma), by Merck Group (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Synergy h1 hybrid multi mode reader, by Agilent Technologies (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions) T4 dna ligase, by Thermo Fisher Scientific (2 mentions) N2 supplement, by Thermo Fisher Scientific (1 mentions) Mars software, by BMG Labtech (1 mentions) Fluostar omega plate reader, by BMG Labtech (1 mentions) Percoll plus, by GE Healthcare (1 mentions) Fetal bovine serum (fbs), by Omega Scientific (1 mentions) Rpmi 1640, by Corning (1 mentions) Cyan flow cytometer, by Beckman Coulter (1 mentions) Summit software, by Beckman Coulter (1 mentions) Cellular reactive oxygen species detection assay kit red fluorescence, by Abcam (1 mentions) Spectramax m5 multi mode microplate reader, by Molecular Devices (1 mentions) N acetyl l cysteine, by Merck Group (1 mentions) Ionomycin, by Alomone (1 mentions)

11 protocols using nunc microwell 96 well optical bottom plates

Replicon Assay for Replication Characteristics

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For the evaluation of the replication characteristics of the p4-cHPstop-SP-IRES-Rluc-Rep and p4-Dualstop-SP-IRES-Rluc-Rep replicons, a replicon assay was performed as described previously (Liu et al., 2013 (link)). For the replicon assays of the UFS mutants, ten thousand BHK-21 cells were seeded into each well of Nunc MicroWell 96-Well Optical-Bottom Plates (Thermo Fisher Scientific, Waltham, Massachusetts) one day prior to transfection. Unless otherwise specified, 100 ng of replicon RNA was transfected per well using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, Massachusetts), and the plates were cultured at 37°C with 5% CO₂. At the indicated time points, the cells in the microplates were lysed with 1× Renilla lysis buffer (Promega, Madison, Wisconsin) and stored at -20°C until use. The Renilla luciferase activity of the collected samples was measured as described previously (Liu et al., 2013 (link)), except that the microplates were directly subjected to the GloMax-96 luminometer.

Liu Z.Y., Li X.F., Jiang T., Deng Y.Q., Ye Q., Zhao H., Yu J.Y, & Qin C.F. (2016). Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization. eLife, 5, e17636.

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Photoconversion and Lineage Tracing of B7.5 Cells

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Photoconversion and lineage tracing were performed as described^{19 (link)}. Fertilized eggs were electroporated with 50 μg Mesp>Kaede:nls to label the B7.5 lineage. Embryos were raised on agarose coated plastic Petri dishes in ASW at 18°C and transferred individually into Nunc™ MicroWell™ 96-Well Optical-Bottom plates (ThermoFisher Scientific, Waltham, MA. Supplier No. 164588) at 15 hpf. Photoconversions were performed using the HC PL FLUOTAR 20×/0.50 objective on Leica Microsystems inverted TCS SP8 X confocal microscope, by shedding 405 nm UV light on ROI continuously for 2 min. Stack scanning of whole TVC lineage were documented at 16, 22.5, 40, 48 and 65 hpf.

Wang W., Niu X., Stuart T., Jullian E., Mauck W., Kelly R.G., Satija R, & Christiaen L. (2019). A single cell transcriptional roadmap for cardiopharyngeal fate diversification. Nature cell biology, 21(6), 674-686.

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Second-Generation RT-QuIC Prion Seeding Assay

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The detection of prion seeding activity in samples was accomplished using the second-generation RT-QuIC assay [9 (link),10 (link)]. All samples were run in quadruplicate. The assay was performed in Nunc™ MicroWell™ 96-Well Optical-Bottom Plates (Thermo Fisher, Prague, Czech Republic) in 100 µL reaction mix for each well (10 mM phosphate buffer, pH 7.4; 300 mM NaCl; 10 µM thioflavin T (ThT); 1 mM EDTA; 0.002% SDS; and 0.1 mg/mL of rSHa PrP (90–231)). For the testing of brain homogenates (BHs), 2 µL of BH were added to 98 µL of reaction volume. BH samples were diluted 5 × 10⁻⁶ up to 5 × 10⁻⁹ in PBS buffer containing 1× N-2 supplement (Gibco, Thermo Fisher, Prague, Czech Republic N-2 supplement, 100×) and 0.1% SDS. CSF samples were tested undiluted or 10 times diluted in PBS (pH 7.4) and 15 µL of CSF sample were added into 85 µL reaction buffer. The reaction was carried out by FLUOstar Omega plate reader (BMG LABTECH GmbH, Ortenberg, Germany), undergoing repeating shaking cycles of 60 s (700 rpm, double orbital) and 60 s rest for 60 h at 55 °C. The fluorescence was measured every 15 min. Each test was analyzed by Mars software (BMG LABTECH GmbH, Ortenberg, Germany).

Moško T., Galušková S., Matěj R., Brůžová M, & Holada K. (2021). Detection of Prions in Brain Homogenates and CSF Samples Using a Second-Generation RT-QuIC Assay: A Useful Tool for Retrospective Analysis of Archived Samples. Pathogens, 10(6), 750.

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Isolation and NET Formation of Neutrophils

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Neutrophil isolation and NET formation were carried out as previously described (39 (link)). Briefly, blood was drawn from patients and normal controls into EDTA-coated tubes and was processed within 3 hours. Red blood cells were first sedimented with Hetastarch (6% hydroxyethyl starch, HES) in 0.9% NaCl solution at 1:4 v/v dilution at 37^oC and then was re-suspended in RPMI1640 (Corning) supplemented with 2% fetal bovine serum (FBS, Omega Scientific). The supernatant was harvested and neutrophils were isolated using Percoll Plus (GE Healthcare) as previously described (39 (link)). Purity of cells was >95% as determined by Wright-Giemsa staining (fig. S1B). For the screening NET assay, NET-bound neutrophil elastase was quantified using an available commercial kit according to manufacturer’s instructions (Cayman chemical). For the validation immunofluorescence assay, neutrophils were resuspended in 2% heat-inactivated FBS and plated at 15,000 cells per well in 96-well optical-bottom plates in triplicates (Nunc MicroWell 96-Well Optical-Bottom Plates, ThermoFisher Scientific). Cells were then stimulated with ionomycin at 4 μM (Sigma-Aldrich) or PMA at 10 and 100 nM (Sigma-Aldrich) for 2.5 hours. Cells were then instantly fixed in 2% paraformaldehyde (PFA) and stained as described below.

Wolach O., Sellar R.S., Martinod K., Cherpokova D., McConkey M., Chappell R.J., Silver A.J., Adams D., Castellano C.A., Schneider R.K., Padera R.F., DeAngelo D.J., Wadleigh M., Steensma D.P., Galinsky I., Stone R.M., Genovese G., McCarroll S.A., Iliadou B., Hultman C., Neuberg D., Mullally A., Wagner D.D, & Ebert B.L. (2018). Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms. Science translational medicine, 10(436), eaan8292.

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Isolation and Stimulation of Murine Neutrophils

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We used a previously published VavCre/Jak2^V617F murine model that results in constitutive heterozygous expression of the Jak2^V617F activating mutation (18 (link)). Peripheral blood was collected from 10 to 12-week-old mice via the retroorbital venous plexus and was processed within 2 hours. Red blood cells were first sedimented with Hetastarch (6% hydroxyethyl starch, HES) in 0.9% NaCl solution at 1:4 v/v dilution at 37^oC. Next, supernatant was collected and subjected to brief hypotonic lysis with sterile water. Neutrophils were isolated by negative selection with magnetic beads according to manufacturer’s instructions (Neutrophil Isolation Kit, mouse, Miltenyi Biotec) and resuspended in 2% heat-inactivated FBS. Neutrophils were plated at 10–15,000 cells per well in 96-well optical-bottom plates in triplicates (Nunc MicroWell 96-Well Optical-Bottom Plates, ThermoFisher Scientific). Cells were then stimulated with ionomycin 4 μM (Sigma-Aldrich) or PMA 10 and 100 nM (Sigma-Aldrich) for 2.5 hours. Cells were then instantly fixed in 2% PFA and stained as described below.

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High-throughput Beta-galactosidase Assay

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NEB® 10-beta cells containing the LacZ fusions of the T4SS + TrbM, TrbO, and TrbP or their respective controls were subcultured in the conditions mentioned in the Bacterial strains and growth conditions section of the supplemental material. All strains were then washed twice with plain LB to remove residual antibiotics and were then resuspended in 200 µL of LB supplemented with CPRG (200 µg/mL). Next, we aliquoted 100 µL of each strain into wells of Nunc MicroWell 96-Well Optical-Bottom Plates from Thermo Scientific™ that were previously filled with 100 µL of LB supplemented with CPRG (200 µg/mL) and six 2-fold serial dilutions of all the strains were performed. Next, the plate was placed inside the Agilent BioTek Synergy H1 Hybrid Multi-Mode Reader pre-warmed to 37 °C, and absorbance readings at 574 nm were taken every 20 minutes for 16 hours. For Supplementary Fig. 8a, the values corresponding to the readings after 16 hours were used. Due to reading overflow, the values corresponding to the readings of 2 hours were used for Supplementary Fig. 8b.

Gordils-Valentin L., Ouyang H., Qian L., Hong J, & Zhu X. (2024). Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer. Communications Biology, 7, 499.

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Doxorubicin cellular accumulation assay

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The cellular accumulation of DOX was ascertained using a Tecan Infinite^® M1000 microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at excitation and emission wavelengths of 485 and 590 nm, respectively. Briefly, the cells were seeded at a 5×10³/well in Nunc™ MicroWell™ 96-well optical-bottom plates (Thermo Fisher Scientific, Inc.) and incubated overnight under standard culture conditions. Media was replaced with culture medium supplemented with various concentrations of papuamine (1, 2 or 5 μM) and doxorubicin (1, 3, 10 and 30 μM) and cultures were incubated for 4 h. The medium was removed and washed twice with PBS, and the residual fluorescence intensity was measured as aforementioned.

KANNO S.I., YOMOGIDA S., TOMIZAWA A., YAMAZAKI H., UKAI K., MANGINDAAN R.E., NAMIKOSHI M, & ISHIKAWA M. (2014). Combined effect of papuamine and doxorubicin in human breast cancer MCF-7 cells. Oncology Letters, 8(2), 547-550.

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Quantifying Reactive Oxygen Levels

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Reactive Oxygen Species (ROS) levels were measured for PEO1^S, PEO4^R, PEA1^S, PEA2^R, PEO14^S, PEO23^R, and FT4 cells using the Cellular Reactive Oxygen Species Detection Assay Kit (Red fluorescence, ab 186027, Abcam, USA). Briefly, 10,000 cells/well of each cell line were plated into Thermo Scientific Nunc MicroWell 96-well optical-bottom plates with polymer base (Cat#165305, Thermo Scientific, USA). Cells were allowed to attach for 48 hours. The manufacturer’s protocol was followed for the ROS assay, and time-resolved fluorescence was monitored at Ex/Em = 520/605 nm with bottom read mode on a SpectraMax M5 Multi-mode microplate reader (Molecular Devices, USA). ROS levels were measured for PEO4 WT and PEO4 KO using a flow cytometry-based assay. Single-cell suspensions were treated with 20 μM DCFDA and the fluorescence (Ex/Em = 485/535 nm) was measured. Flow cytometry was performed at the University of Illinois at Chicago RRC facility using CyAn flow cytometer (Beckman Coulter Inc., Fullerton, CA). All data were analyzed by Summit software (Beckman Coulter Inc., Fullerton, CA). N-Acetyl-L-cysteine (NAC) was purchased from Sigma (MO, USA), (#1009005).

Huang D., Chowdhury S., Wang H., Savage S.R., Ivey R.G., Kennedy J.J., Whiteaker J.R., Lin C., Hou X., Oberg A.L., Larson M.C., Eskandari N., Delisi D.A., Gentile S., Huntoon C.J., Voytovich U.J., Shire Z.J., Yu Q., Gygi S.P., Hoofnagle A.N., Herbert Z.T., Lorentzen T.D., Calinawan A., Karnitz L.M., Weroha S.J., Kaufmann S.H., Zhang B., Wang P., Birrer M.J, & Paulovich A.G. (2021). Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer. Cell Reports Medicine, 2(12), 100471.

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VZV Glycoprotein Fusion Assay

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Tacrolimus (FK506) (Selleckchem), pimecrolimus (Selleckchem), sirolimus (Selleckchem), and ionomycin (Alomone labs) were all dissolved in DMSO (Sigma Aldrich). Activity of the compounds on VZV gB/gH-gL mediated cell fusion were evaluated by stable reporter fusion assay as described previously [28 (link)]. Briefly, CHO-DSP1 or MeWo-DSP1 cells transfected with equal quantities of pCAGGS-gB, pME18S-gH[TL], and pcDNA3.1-gL plasmids using Lipofectamine 2000 were harvested at 6 hrs post-transfection, and mixed with MeWo-DSP2 cells in the presence of various concentrations of the compounds prepared in two-fold serial dilutions, ranging from 10 μM to 1.25 μM. Co-culture of cells were seeded into Nunc MicroWell 96-well Optical-Bottom Plates (ThermoFisher) and incubated for 48 hrs. The activity of Renilla luciferase was read immediately after adding substrate h-Coelenterazine (Nanolight Technology). Transfection with only vehicle plasmids pcDNA3.1 (+) and pME18S, or pcDNA3.1 (+), pME18S and pCAGGS-gB served as negative control. Cell viability was measured using CellTiter-Glo Luminescent substrate (Promega). Luminescence signal was recorded using Synergy H1 Hybrid Multi-Mode Reader (BioTek). Experiments were performed at least in triplicate.

Zhou M., Kamarshi V., Arvin A.M, & Oliver S.L. (2020). Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion. PLoS Pathogens, 16(11), e1009022.

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Recombinant Protein Expression and Purification

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Synthetic DNA oligonucleotides and gBlocks were purchased from Integrated DNA Technologies (IDT) (Supplementary Table I). Plastic consumables, restriction endonucleases, Taq polymerase, Phusion polymerase, T4 DNA ligase, deoxynucleotides, DH10B Escherichia coli., pBAD/His B plasmid, pcDNA3.1(+) plasmid, Bacterial Protein Extraction Reagent (B-PER), Penicillin–Streptomycin, Fetal Bovine Serum (FBS), TurboFect, and GeneJET gel and plasmid purification kits were purchased from ThermoFisher. Agarose, MnCl₂·4H₂O_, D-glucose, ampicillin, L-arabinose, Hank’s balanced salt solution (HBSS), Dulbecco’s Modified Eagle Medium (DMEM), TryplE Express, and LB Lennox media were purchased from Fisher Scientific. Nickel nitrilotriacetic acid (NTA) immobilized metal affinity chromatography protein purification beads were purchased from G-BioSciences. Ethidium bromide and polymerase chain reaction (PCR) instruments (T100 Thermal Cycler) were purchased from BioRad. Gibson Assembly reagent was purchased from New England Biolabs. QuikChange mutagenesis kits were purchased from Agilent Technologies. Nunc MicroWell 96-Well Optical-Bottom Plates (catalog #265301) were purchased from ThermoFisher. Molecular weight cutoff filters were purchased from Millipore-Sigma. Sequencing was performed by the Molecular Biology Services Unit (MBSU) at the University of Alberta.

Zarowny L., Clavel D., Johannson R., Duarte K., Depernet H., Dupuy J., Baker H., Brown A., Royant A, & Campbell R.E. (2022). Cyan fluorescent proteins derived from mNeonGreen. Protein Engineering, Design and Selection, 35, gzac004.

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