384 well plate

Manufactured by Corning

Sourced in United States, United Kingdom, China

The 384-well plates are a type of laboratory equipment designed for high-throughput screening and assay applications. These plates feature an array of 384 individual wells, each with a small volume capacity, allowing for efficient processing of multiple samples simultaneously. The 384-well format is commonly used in various scientific disciplines, such as cell-based assays, biochemical assays, and drug discovery research.

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Close spelling variants of this product

Low volume non-binding surface 384-well plates High-binding 384-well polystyrene plates 384-well flat-bottomed plates Poly-L-lysine–coated 384-well plates 384-well ELISA plates Falcon Corning black 384 well plates Cell-BIND® 384 well plates Black 384-well low-volume plates Ultra-low attachment 384-well plates Corning 384-well format 3D spheroid culture plates

124 protocols using 384 well plate

High-Throughput Screening of Trypanocidal Compounds

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Compounds exhibiting activity of >50% in duplicate at a final concentration of 18.3 μM, with associated activity of >50% at 9.2 μM were selected for retest in dose response to determine IC₅₀ values. A separate group was also retested, with >50% activity exhibited at 18.3 μM, with single point, or no corresponding activity at 9.2 μM. Some compounds appeared to come out of solution in water at the highest dose of 18.3 μM, the initial dose for retesting compounds was reduced to 9.2 μM. Compounds were cherry-picked from 5 mM stocks in 100% DMSO and diluted in DMSO, to a working concentration of 2.5 mM. Compounds were diluted into 15 doses in 384-well plates (Axygen, USA), before dilution into water prior to addition in the assay. The final assay concentrations ranged from 9.2 μM to 0.0002 μM. Compounds were retested in two independent experiments. Image capture and analysis was undertaken on the Operetta image-based system, at 20× magnification.
Compounds that were considered active had an IC₅₀ value of <10 μM, and if the IC₅₀ could be determined against 3T3 cells, with a SI >10. If the IC₅₀ value against 3T3 cells could not be determined, the SI was calculated by dividing the highest dose of 3T3 cells in the assay screened by the IC₅₀ value of the compound against T. cruzi amastigotes.

Sykes M.L, & Avery V.M. (2015). Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes. International Journal for Parasitology: Drugs and Drug Resistance, 5(3), 215-228.

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Gene Expression Analysis by RT-qPCR

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Total RNA was extracted from tissues or cultured cells using TRIzol (Invitrogen) according the manufacturer’s protocol. RNA (1 μg) was used for reverse transcription with ReverTra Ace (Toyobo) using random hexameric oligos at 42°C for 1 hour, and the complementary DNA products were appropriately diluted with Milli-Q water. To rule out contamination, additional reverse transcription reactions containing no RNA were prepared as the negative controls for real-time PCR. Quantitative real-time PCR was carried out using a SYBR qPCR Mix (Toyobo) in a 10-μl reaction volume with a LightCycler 480 II (Roche) in 384-well plates (Axygen). Each reaction was repeated in three wells. We confirmed the specificity of the real-time PCR products by melting curve analysis. All gene expression levels were normalized to β-actin.

Fang Z., Zhou L., Jiang S., Cao L, & Yu L. (2015). UNC50 Prompts G1/S Transition and Proliferation in HCC by Regulation of Epidermal Growth Factor Receptor Trafficking. PLoS ONE, 10(3), e0119338.

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Genetic Polymorphisms in Zn-Related Proteins

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Five SNPs in five genes coding Zn-related proteins were analyzed: rs1799750 in MMP-1, rs243865 in MMP-2, rs11568818 in MMP-7, rs2252070 in MMP-13 and rs28366003 in MT2A. Genotyping was performed by Real-Time PCR using Taqman probes. The rs243865 in MMP-2, rs11568818 in MMP-7, rs2252070 in MMP-13 and rs28366003 in MT2A were analyzed using pre-designed Genotyping Assays. For analysis of rs1799750 in MMP-1 a customized assay was used.
The reaction mix for analysis of each sample consisted of GoTaq^® Probe qPCR Master Mix (Promega, USA), TaqMan Genotyping Assay x 40 (Applied Biosystems, USA) and deionized water in accordance to TaqMan Genotyping Assay’s manufacturer’s instructions. The reaction mix and sample DNA were placed in 384 well plates (Axygen, USA) and Real-Time PCR reactions were performed on a LightCycler^® 480 (Real-Time PCR System, Roche Diagnostics, USA). For genotyping data analysis LightCycler480 Basic SoftwareVersion1.5 was used (Roche Diagnostics, USA).

Białkowska K., Marciniak W., Muszyńska M., Baszuk P., Gupta S., Jaworska-Bieniek K., Sukiennicki G., Durda K., Gromowski T., Prajzendanc K., Cybulski C., Huzarski T., Gronwald J., Dębniak T., Scott R.J., Lubiński J, & Jakubowska A. (2018). Association of zinc level and polymorphism in MMP-7 gene with prostate cancer in Polish population. PLoS ONE, 13(7), e0201065.

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qRT-PCR for Gene Expression Analysis

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qRT-PCR reactions were performed in 384 well plates (Axygen) and contained 1 μl diluted cDNA reverse transcribed from total RNA (equivalent to 0.5 μg) in nuclease-free H₂O (1:5), 50 nM forward and reverse primer, 1x final concentration of SYBR green PCR master mix (Applied Biosystems) and nuclease-free H2O (total volume of 10 μl). Reactions were performed using a ViiA7 real-time PCR system (Life Technologies). Cycling conditions were 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min followed by a primer-template dissociation step. Gene expression was normalized to 7SL mRNA levels using the comparative CT (CT) method. The primer sequences for the human genes are in Supplemental Table S1.

Paquet N., Adams M.N., Leong V., Ashton N.W., Touma C., Gamsjaeger R., Cubeddu L., Beard S., Burgess J.T., Bolderson E., O'Byrne K.J, & Richard D.J. (2015). hSSB1 (NABP2/ OBFC2B) is required for the repair of 8-oxo-guanine by the hOGG1-mediated base excision repair pathway. Nucleic Acids Research, 43(18), 8817-8829.

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Protein Microarray Fabrication

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The protein was mixed with sample buffer (Capital-bio, Beijing, China), then put into 384 well plates (Axygen, CA), and made with microarray printing device (Capital-bio, Beijing, China). Polymer 3D substrate (Capital-bio, Beijing, China) was selected, for each protein by three times of repetition. The proteins in the microarray after printed were quality checked by antibodies.

Chen P., Zeng Z., & Du H. (2020). Establishment and Validation of a Drug-Target Microarray for SARS-CoV-2.

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T-cell Activation Assay with Oclacitinib

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Human whole blood was collected in heparin sulfate tubes. Whole blood (75 μL per well) was plated in 384-well plates (Costar Corning) containing vehicle control or oclacitinib (0.000143–37.5 μm). Treated whole blood was then stimulated with mouse anti-human CD3 (1 μg/mL; BD Biosciences), mouse anti-human CD28 (10 ng/mL; BD Biosciences), and recombinant human IL-12 (10 ng/mL; R&D Systems) overnight at 37 °C, 5% CO₂. Whole blood was spun to separate plasma, and 20 μL of plasma was collected. Interferon gamma levels were quantitated in plasma using Meso IFN-gamma 384-well plate kit and a Meso Scale Discovery SECTOR Imager 6000 (Rockville, MD, USA) following the manufacturer's instructions. Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Gonzales A.J., Bowman J.W., Fici G.J., Zhang M., Mann D.W, & Mitton-Fry M. (2014). Oclacitinib (APOQUEL®) is a novel Janus kinase inhibitor with activity against cytokines involved in allergy. Journal of Veterinary Pharmacology and Therapeutics, 37(4), 317-324.

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DFHBI-1T Fluorescence RNA Detection

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Measurements were performed in 384-well plates (Costar) with sample volumes of 50 μl containing 100 nM RNA, 500 nM DFHBI-1T, 40 mM Hepes, 50 mM KCl, and 5 mM MgCl₂. DFHBI-1T was purchased from Lucerna Technologies. Fluorescence was monitored using a CLARIOstar Plus multi-mode microplate reader (BMG LABTECH) set at a constant temperature of 25°C. For the addition of RNA keys, the plates were removed from the plate reader, individual keys were added and mixed in the laboratory, and then the plates were reinserted into the machine for continued measurements. Excitation of DFHBI-1T was performed at 470 ± 10 nm and emission was recorded at 505 ± 10 nm for the time point measurements. For the excitation spectral scans, emission was recorded at 505 nm while excited from 420 to 480 nm. For the emission spectral scans, 470 nm was used for excitation and emission was recorded from 500 to 550 nm.

Vallina N.S., McRae E.K., Geary C, & Andersen E.S. (2024). An RNA origami robot that traps and releases a fluorescent aptamer. Science Advances, 10(12), eadk1250.

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SARS-CoV-2 Spike Protein Binding Assay

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Recombinant spike protein 1 (S1) of SARS-CoV-2 (2 μg/ml, Sino Biological, Beijing, China) were coated on 384-well plates (Corning Costar), and incubated at 4 °C ovnernight. Then the plates were blocked with the blocking buffer (PBS with 5% BSA) at 37 °C for 1 h, and subsequently added with serially diluted monoclonal antibodies (10-fold dilutions, from 10 μg/ml to 10 pg/ml). After 30 min incubation at 37 °C, the plates were washed 5 times and incubated with goat anti-human IgG (H + L) antibody conjugated with ALP (Thermo Fisher, a18808, 1:5000) for 30 min at 37 °C. For the quantification of bound IgG, 1 mg/ml PNPP (Thermo Fisher) was added and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).

Li S., Wang W., Li T., Han X., Hu C., Wang Y., Shen M., Du L., Nai Y., Wang J, & Jin A. (2020). Immune characteristics analysis reveals two key inflammatory factors correlated to the expressions of SARS-CoV-2 S1-specific antibodies. Genes & Diseases, 9(2), 522-530.

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E. coli Toxicity Profiling Assay

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The SUC standard and disinfected samples were evaluated for toxicity using an E. coli whole-cell microarray assay based on toxicological genomics. The whole-cell assay library, including 110 genes with GFP reporter (Supplementary Table S1) of the E. coli K-12 MG1655 strain, was used for toxicity assay. The 110 genes chosen are involved in different cellular stress responses, including oxidative stress, DNA stress, protein stress, general stress, and membrane stress. Each fusion was expressed by a lowcopy plasmid pUA66 (Supplementary Figure S1), which contained a kanamycin resistance gene and a fast-folding GFPmut2, allowing for real-time measurement of changes in gene expression levels (Du et al., 2017; (link)Lan et al., 2018) (link). The E. coli strains were cultivated in LB medium with 25 mg/L kanamycin in 384-well plates (CoStar, Bethesda, MD, USA) at 37 °C in dark to avoid GFP photobleaching till the early exponential growth stage (characterized by an OD 600 value of 0.1-0.3). After sample addition, the plate was placed in a microplate reader (Cytation, Neo2, BioTek, USA) for cell growth measurement (at a wavelength of 600 nm) and fluorescence data acquisition (at excitation and emission wavelengths of 485 and 528 nm, respectively) at 5-min intervals for 2 h at 37 °C. Three replicate samples were used for each test.

Zhai Y., Bai D., Yang H., Li X., Zhu D., Cao X., Ma H., Li X., & Zheng X. (2021). Hazardous Effects of Sucralose and Its Disinfection Byproducts Identified From an E. coli Whole-Cell Array Analysis. Frontiers in Environmental Science, 9.

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Fluorescence Polarization Assay for Nrf2-Keap1 Interaction

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The fluorescence polarisation (FP) assays were performed in a similar manner as previously described³⁰ (link). The experiments were performed briefly on a Synergy H4 microplate reader (BioTek, USA) using the 485 nm excitation and 535 nm emission filters for the FITC. The plates used for the FP measurements were Corning 384-well plates (product #3575). The assay buffer used in this assay consisted of 10 mM HEPES buffer (pH 7.4), 50 mM EDTA, 150 mM NaCl and 0.005% Tween-20. Each well was loaded with a total 40 μL assay solution containing 20 nM FITC-9mer Nrf2 peptide amide (FITC-LDEETGEFL-NH₂, 10 μL), 400 nM Keap1 Kelch domain protein (10 μL), and an inhibitor sample at different concentrations (10 μL). Afterwards, the plate was covered and shaken for 30 min at room temperature before FP measurements. Then, the parallel and perpendicular fluorescence intensity (F_ǁ and F^⊥) relative to the linearly polarised excitation light was measured and the FP was determined. Each experiment was replicated three times. The IC₅₀ values of the tested compound were ascertained from the plot of %inhibition against inhibitor concentration using GraphPad Prism 7.0 (Graphpad Software, Inc., USA).

Sun Y., Zheng L., Yang B., Ge S., Li Q., Zhang M., Shen S, & Ying Y. (2022). Design, synthesis and evaluation of novel small molecules acting as Keap1-Nrf2 protein-protein interaction inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 2575-2588.

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