Synergy h1 plate

Manufactured by Agilent Technologies

Sourced in United States

The Synergy H1 plate is a multi-mode microplate reader designed for a variety of laboratory applications. It is capable of performing absorbance, fluorescence, and luminescence measurements. The device offers a compact and flexible design to accommodate different microplate formats.

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Lab products found in correlation

Mtt assay kit, by Promega (1 mentions) Nad nadh glo, by Promega (1 mentions) Nadp nadph glo assays kit, by Promega (1 mentions) Vortemp 56, by Labnet (1 mentions) Dual glo luciferase assay kit, by Promega (1 mentions) Pg13 luc plasmid, by Addgene (1 mentions) Prl tk plasmid, by Promega (1 mentions) Laurdan, by Thermo Fisher Scientific (1 mentions) Black 96 well plate, by Greiner (1 mentions) Celltiter glo 2.0 reagent, by Promega (1 mentions)

11 protocols using synergy h1 plate

Biofilm Pellicle Formation Assay

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Biofilm pellicle
formation assays were performed as described previously.^{16 (link)} For the initial screening assay, compounds were
tested at the concentrations of 50, 25, 10, 5, and 2.5 μM for
inhibition of biofilm pellicle formation, which was evaluated by eye
following 3 weeks of incubation under low oxygen and 2 weeks of incubation
under aerated conditions. For the assays involving the determination
of the IC₅₀ for biofilm inhibition by C10, 17h, and 17j, Sauton’s medium was inoculated
with stationary-phase Mtb with and without two-fold
dilutions of C10 ranging from 0.39–100 to 0.08–20
μM for the more potent compounds, 17h and 17j; untreated controls were included in every row of the
assay. The plates were sealed in airtight containers and incubated
at 37 °C with 5% CO₂. After 3 weeks, the lid of the
container was removed, and the plates were incubated for an additional
3 weeks. At the end of the 6-week treatment, the plates were photographed
and the OD₆₀₀ through the pellicle at the air/liquid interface
in each well was quantified using a BioTek Synergy H1 Plate reader
to determine pellicle density. In control wells containing tyloxapol,
the procedure was the same but 0.05% tyloxapol was added to each well.

Sarkar S., Mayer Bridwell A.E., Good J.A., Wang E.R., McKee S.R., Valenta J., Harrison G.A., Flentie K.N., Henry F.L., Wixe T., Demirel P., Vagolu S.K., Chatagnon J., Machelart A., Brodin P., Tønjum T., Stallings C.L, & Almqvist F. (2023). Design, Synthesis, and Evaluation of Novel Δ2-Thiazolino 2-Pyridone Derivatives That Potentiate Isoniazid Activity in an Isoniazid-Resistant Mycobacterium tuberculosis Mutant. Journal of Medicinal Chemistry, 66(16), 11056-11077.

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Herd Immunity in Liquid Bacterial Cultures

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Herd immunity in a liquid culture was tested in 200

μ l

of LB broth supplemented with 25

μ g / m l

chloramphenicol in Nunclon flat bottom 96 well plate in a Bio-Tek Synergy H1 Plate reader. Bacterial cultures were diluted 1 in 1000 and mixed in the following ratios of resistant to susceptible cells: 50:50, 75:25, 87.5:12.5, 93.75:6.25, 96.88:3.13, 98.44:1.56, 99.22:0.78, 99.61:0.39, 99.8:0.2, 99.9:0.1, 99.95:0.05, 100:0 %. T7 phage was added at a multiplicity of infection (MOI) of

\approx 10^{- 4}

(

\approx 50

plaque forming units (

p f u

) per culture) to resemble an epidemic initiated by the burst size from one infected cell and the cultures were monitored at an optical density 600 nm for 18 hours post inoculation (

h p i

). Each population composition was replicated 16 times. Herd immunity in diluted LB was measured in LB broth mixed with 1X M9 salts in ratios 1:1 (50% LB) and 1:4 (20% LB) using the same protocol as for 100% LB broth. Each population composition was replicated 18 times.

Payne P., Geyrhofer L., Barton N.H, & Bollback J.P. (2018). CRISPR-based herd immunity can limit phage epidemics in bacterial populations. eLife, 7, e32035.

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MTT Assay Using Promega's Kit

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An MTT assay was performed using Promega’s MTT assay kit (Promega, WI, USA) according to the manufacturer’s protocol. Assays were read at 570 nm using a BioTek Synergy H1 plate reader.

Ahmad I.M., Dafferner A.J., Salloom R.J, & Abdalla M.Y. (2023). Heme Oxygenase-1 Inhibition Modulates Autophagy and Augments Arsenic Trioxide Cytotoxicity in Pancreatic Cancer Cells. Biomedicines, 11(9), 2580.

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Quantification of NAD(P)(H) Levels

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NAD⁺, NADP⁺, NADH, and NADPH levels, as well as NAD+/NADH and NADP⁺/NADPH ratios were examined using the NAD/NADH-Glo™ or NADP/NADPH-Glo™ Assays kit (Promega, USA) according to the manufacturer’s protocols. Briefly, 10.000 cells were plated in a 96 well plate 24 h before treatment. Cells were lysed followed by measuring NAD(P)⁺ or NAD(P)H luminescence signal separately by using BioTek Synergy H1 plate reader (Biotek, USA). The ratio of NAD+/NADH and NADP+/NADPH were calculated based on the instruction of the kits.

Xiang K., Kalthoff C., Münch C., Jendrossek V, & Matschke J. (2022). Accumulation of oncometabolite D-2-Hydroxyglutarate by SLC25A1 inhibition: A metabolic strategy for induction of HR-ness and radiosensitivity. Cell Death & Disease, 13(7), 641.

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Growth Curve Assay for Historical and Emergent Strains

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Growth curves for the four historical and four emergent clade strains were performed in THY or C medium as previously described (62 (link)). Briefly, overnight cultures grown at 37°C and 5% CO₂ were used to inoculate (1/50 volume) prewarmed medium and distributed to a 96-well microtiter plate. Growth was monitored in a BioTek Synergy H1 plate reader, and readings were obtained every 30 min following shaking to redistribute cells in individual wells. Individual strains were assayed in technical quadruplicate and in biological triplicate on two independent days. Milk plate assays were performed in technical quadruplicate as previously described (39 (link)).

DebRoy S., Sanson M., Shah B., Regmi S., Vega L.A., Odo C., Sahasrabhojane P., McGeer A., Tyrrell G.J., Fittipaldi N., Shelburne S.A, & Flores A.R. (2021). Population Genomics of emm4 Group A Streptococcus Reveals Progressive Replacement with a Hypervirulent Clone in North America. mSystems, 6(4), e00495-21.

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Quantifying Bacterial Gene Expression

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Plasmids containing the ompF-sfgfp reporter, p_Lux-MicF, or controls were transformed into E. coli MG1655, plated onto LB + Agar plates containing 34 μg/mL chloramphenicol and 100 μg/mL carbenicillin, and incubated overnight at 37°C. Three colonies from each condition were inoculated into 300 μL of LB with corresponding antibiotics in a 2 mL 96-well block (Costar 3961) sealed with a breath-easier membrane and grown for 17 hours overnight at 37°C while shaking at 100 rpm (Labnet Vortemp 56). Four microliters of the overnight culture were added to 296 μL of MOPS media that was pre-warmed at 37°C for 30 minutes. The cultures were grown under the same conditions as above for approximately 2.5–3 hours until an OD600 of 0.35–0.4 was reached. Cultures were diluted a second time using pre-warmed MOPS into a 96-well plate (Corning 3631) to reach an OD600 of approximately 0.015. AHL was added to each well at a final concentration of 2 nM. CPP-PNAs were added to each well at final concentrations indicated in the figures. The plate was sealed with a breathe-easy membrane and placed on a Biotek Synergy H1 plate reader. The temperature was controlled at 37°C with continuous double-orbital shaking (425 cpm) for two hours. OD600 and SFGFP fluorescence was measured from the bottom of the plate every ten minutes (485 nm excitation, 520 nm emission, gain 80).

Tsai M.J., Zambrano R.A., Susas J.L., Silva L, & Takahashi M.K. (2023). Identifying antisense oligonucleotides to disrupt small RNA regulated antibiotic resistance via a cell-free transcription-translation platform. bioRxiv.

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Evaluating Axin and P53 Peptide Analogues

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For evaluation of Axin peptide analogues, DLD-1 cells were cultured in 96-well flat bottom plates up to 70–80% confluency and were transfected with 200 ng M50 TOPFlash plasmid (a gift from Professor Raymond Habas, Temple Biology) and 50 ng pRL-TK plasmid (Promega). 12 h after transfection, cells were treated with DMSO vehicle or 15 μM of Axin peptide analogues for another 24 h. Luciferase activity was measured using the Dual-Glo luciferase assay kit (Promega) on a Biotek Synergy H1 plate reader using the Gen5 software. Data were processed as firefly: Renilla luciferase ratio for each control or treatment group, and were further normalized against the vehicle control group (100%) as the Relative Activities.
For evaluation of P53 peptide analogues, HCT116 wt cells were transfected with 200 ng PG13-luc plasmid (a gift from Bert Vogelstein (Addgene plasmid #16442; http://n2t.net/addgene:16442; RRID:Addgene_16442)) and 50 ng pRL-TK plasmid. At about 12 h after transfection, the cells were treated with solvent vehicle or 15 μM of each p53 peptide analogue for an additional 24 h. The follow-up luciferase activity measurement and data processing were completed in the same manner as mentioned above.

Islam M.S., Junod S.L., Zhang S., Buuh Z.Y., Guan Y., Zhao M., Kaneria K.H., Kafley P., Cohen C., Maloney R., Lyu Z., Voelz V.A., Yang W, & Wang R.E. (2022). Unprotected peptide macrocyclization and stapling via a fluorine-thiol displacement reaction. Nature Communications, 13, 350.

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ATP Leak Assay for Antibiotic Susceptibility

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ATP leak assay was performed using the Enhanced ATP Assay Kit of Beyotime (S0027) according to the manufacturer's manual. Briefly, the log-phase culture of A. baumannii YQ4 was prepared, washed, and suspended in PBS as above. This suspension was exposed to 16 μg/ml of COG1410, 16 μg/ml of polymyxin B, and 32 μg/ml of tigecycline, respectively, and incubated at 37°C for 30 min. The supernatant was harvested and used for the measurement of ATP levels. In total, 100 μl of supernatant was mixed with 100 μl of working solution, and the chemiluminescence was measured by using a Biotek Synergy H1 plate reader. The untreated sample was set as a negative control. The experiments were performed in triplicate.

Wang B., Zhang F.W., Wang W.X., Zhao Y.Y., Sun S.Y., Yu J.H., Vitek M.P., Li G.F., Ma R., Wang S., Hu Z, & Chen W. (2022). Apolipoprotein E mimetic peptide COG1410 combats pandrug-resistant Acinetobacter baumannii. Frontiers in Microbiology, 13, 934765.

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Measuring Membrane Fluidity Using Laurdan

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Overnight cultures were subcultured 1:50 in fresh LBg (LB with 0.2% glucose) and grown to mid-log phase (OD₆₀₀ 0.4–0.6). Laurdan (Invitrogen) was added to a final concentration of 10 μM and incubated at 37°C with rotation for 30 minutes. Cells were harvested by centrifugation, washed three times, and resuspended in prewarmed PBSg (PBS with 0.2% glucose). Cells (200 μL) were transferred to a black 96-well plate (Greiner) and a monitored (ex360/em450 and 500 nm) on a BioTek Synergy H1 plate reader. Baseline fluorescence was recorded for five minutes prior to addition of compound. Fluorescence was recorded for 25 additional minutes. Laurdan generalized polarization (GP) was calculated: GP = (I₄₆₀- I₅₀₀)/ (I₄₆₀+ I₅₀₀).

Dombach J.L., Quintana J.L., Nagy T.A., Wan C., Crooks A.L., Yu H., Su C.C., Yu E.W., Shen J, & Detweiler C.S. (2020). A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids. PLoS Pathogens, 16(12), e1009119.

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Intracellular ATP Measurement Protocol

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Intracellular ATP levels were measured using a BacTiter-Glo microbial cell viability assay (Promega) according to the manufacturer’s instructions. Mid-log-phase cells (100 μl) were added to DMSO, chloramphenicol (32 μg/ml) (81 (link)), or 2 μl of compound in a black polystyrene 96-well plate (Greiner; 655076) and incubated for 10 or 25 min at 37°C with agitation. Reagent (100 μl) was added, and samples were incubated in the dark with agitation for 5 min. Luminescence was read on a BioTek Synergy H1 plate reader.

Dombach J.L., Quintana J.L, & Detweiler C.S. (2021). Staphylococcal Bacterial Persister Cells, Biofilms, and Intracellular Infection Are Disrupted by JD1, a Membrane-Damaging Small Molecule. mBio, 12(5), e01801-21.

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