96 well plate

Manufactured by Techno Plastic Products

Sourced in Switzerland, Japan

The 96-well plates are a standard laboratory equipment used for various experimental and analytical purposes. They provide a multi-well format for conducting parallel experiments or assays. The plates typically have 96 individual wells arranged in a 8x12 grid pattern, allowing for the simultaneous processing of multiple samples or reactions.

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Lab products found in correlation

Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions) Leibovitz l 15 medium, by Harvard Bioscience (1 mentions) Dulbecco s phosphate buffered saline, by Merck Group (1 mentions) Bmg fluostar optima, by BMG Labtech (1 mentions) Alizarin red staining solution, by Merck Group (1 mentions) Microplate reader, by Bio-Rad (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Fluorescein, by Merck Group (1 mentions) Trolox, by Merck Group (1 mentions) Fluostar optima, by BMG Labtech (1 mentions) 3h thymidine, by GE Healthcare (1 mentions) Cellometer mini automated cell counter, by Revvity (1 mentions) Biomek fx laboratory automation workstation, by Beckman Coulter (1 mentions) Sodium deoxycholate, by Merck Group (1 mentions) High glucose dmem, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

29 protocols using 96 well plate

Neutralization Assay for Tick-Borne Encephalitis Virus

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Sera were heat-inactivated for 30 min. at 56 °C and subsequently diluted 1:8 in Leibovitz L-15 medium (Biochrom AG, Berlin, Germany) supplemented with 5% FBS (Seraglob, Schaffhausen, Switzerland). Further fivefold dilutions were made in duplicates in a 96-well plate (TPP Techno Plastic Products, Trasadingen, Switzerland) in a total volume of 50µL. One hundred TCID₅₀ TBEV (Hypr, provided by Daniel Růžek, University of South Bohemia, České Budějovice, Czech Republic) were added in equal volume. Plates were incubated overnight at 4 °C and subsequently at 37 °C for 1 h without CO₂. Porcine kidney stable (PS) cells (provided by Daniel Růžek, University of South Bohemia, České Budějovice, Czech Republic) were then added to each well (15,000 cells/well in 100µL), and plates further incubated at 37 °C without CO₂. On day 3, neutral red dye in Dulbecco’s phosphate-buffered saline (Sigma Aldrich, Buchs SG, Switzerland) was added to each well at a final concentration of 0.000165%. On day 5, the liquid was removed, and the presence or absence of neutral-red-stained cells used to assess virus-induced cytopathic effect (CPE) and neutralizing capacity reported as the geometric mean titer (GMT) of two replicates.
In all neutralization tests, neutralization titer was defined as the reciprocal dilution resulting in at least 50% virus neutralization.

Kohler P., Jonsdottir H.R., Risch L., Vernazza P., Ackermann-Gäumann R, & Kahlert C.R. (2021). No neutralizing effect of pre-existing tick-borne encephalitis virus antibodies against severe acute respiratory syndrome coronavirus-2: a prospective healthcare worker study. Scientific Reports, 11, 24198.

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Evaluating Metabolic Activity with SKR

Cited 1 time

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Metabolic activity assays (MTT) were performed as reported previously [49 (link)] to evaluate changes in the metabolic activity of cells after treatment with SKR. Solutions of SKR (from 0.5

μ

M to 20

μ

M) were added to cells in a 96-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland) to examine the SKR effect on metabolic activity based on MTT assay and to determine SKR concentrations for further experiments. Of note, IC50 values can be only extrapolated, because of the relatively low inhibition rate of SKR after the shorter incubation period. After 24 and 48 h, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) from a stock solution (5 mg/mL) was added to the cells in a 96-well plate. The reaction was stopped after 4 h incubation and the insoluble formazan was dissolved by the addition of SDS at a final concentration of 3.3%. The absorbance of metabolized formazan (λ = 584 nm) was measured using a BMG FLUOstar Optima (BMG Labtech GmbH, Offenburg, Germany). Results were evaluated as the ratio of the absorbance of the treated sample to the untreated matching control. The experimental groups were compared with the matching control groups (normoxic control or hypoxic control).

Babinčák M., Jendželovský R., Košuth J., Majerník M., Vargová J., Mikulášek K., Zdráhal Z, & Fedoročko P. (2021). Death Receptor 5 (TNFRSF10B) Is Upregulated and TRAIL Resistance Is Reversed in Hypoxia and Normoxia in Colorectal Cancer Cell Lines after Treatment with Skyrin, the Active Metabolite of Hypericum spp.. Cancers, 13(7), 1646.

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Fibroblast Cell Line Cytotoxicity Assay

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The experiments were performed on a FELCH-104 stable cell line from BioloT Ltd. (Saint-Petersburg, Russia), which presents a culture of fibroblast-like cells derived from an 8-week human embryo. The cell culture was kept at 37°С under a 5% CO₂ atmosphere in a DMEM medium containing L-glutamine, 1 g/L glucose, 10% embryonic bovine serum, and .5% gentamicin. We waited till a monolayer has been formed having investigated the preparation under inverted microscope each 24 hours after seeding the cells. For assessing NP cytotoxicity, cells were seeded in a 96-well plate (ТРР Techno Plastic Products AG, Trasadingen, Switzerland), 70 000 cells per well in 100 mcL medium, and maintained under standard conditions for 48 hours until a monolayer was obtained. Then suspensions of CuO and SeO nanoparticles (diluted to a needed concentration with DMEM medium) were added to the wells and incubated for 24 hours under standard conditions before performing an ATP assay. The final concentration of each NP type in the medium was 25-50-100 mcg/mcL. Nanoparticles (ether of one species or their combination) were added to the culture medium in these concentrations.

Panov V., Bushueva T., Minigalieva I., Naumova A., Shur V., Shishkina E., Sutunkova M., Gurviсh V., Privalova L, & Katsnelson B. (2021). New Data on Variously Directed Dose-Response Relationships and the Combined Action Types for Different Outcomes of in Vitro Nanoparticle Cytotoxicity. Dose-Response, 19(4), 15593258211052420.

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NHDF Proliferation on Collagen/Chitosan Scaffolds

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All scaffolds were pre-incubated 1 h before NHDF seeding in 150 μl of culture medium in 96 well plate (Techno Plastic Products, Switzerland) to ensure treminated swelling and volume increase. After NHDF passage and counting in Bürker chamber, culture medium was aspirated from the scaffolds and 20 000 NHDF were seeded on each scaffold in 150 μl of culture medium containing only 0.5% FBS (previously used 10% FBS) to detect the possible effect of FGF2-STAB^® in scaffolds. NHDF were cultured for 1 and 3 days followed by metabolic activity assay and by microscopic evaluation. Scaffolds Collagen/Chitosan represented untreated control and scaffolds Collagen/Chitosan enriched by SeNPs and/or by FGF2-STAB^® represented tested samples.

Muchová J., Hearnden V., Michlovská L., Vištejnová L., Zavaďáková A., Šmerková K., Kočiová S., Adam V., Kopel P, & Vojtová L. (2021). Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds: in vitro and ex ovo evaluation. Journal of Nanobiotechnology, 19, 103.

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MC3T3-E1 Osteoblast Mineralization

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ECM mineralization by MC3T3-E1 pre-osteoblasts attached to the scaffolds was analyzed after 7, 14, 21, and 28 days of culture. To determine mineralization, one part (1/8th) of the cell/scaffold construct was washed with PBS, and fixed in 4% glutaraldehyde for 15 min at 37°C. Fixed constructs were incubated in 40 mM Alizarin Red staining solution (Merck, Darmstadt, Germany), pH 4.3, at room temperature for 30 min, and washed extensively with deionized water to remove the unreacted dye. Optical images were taken using a stereomicroscope. For quantitative mineralization analysis, the red-stained mineralized nodules were dissolved with 5% sodium dodecyl sulfate (Merck) in 0.5 N HCL at room temperature under shaking. Then, 100 µl of the solution was added per well of a 96-well plate (Techno Plastic Products, Trasadingen, Switzerland) to measure the absorbance at 405 nm with a microplate reader (BioRad Laboratories Inc., Veenendaal, Netherlands). Scaffolds were air-dried at room temperature for 24 h and weighed. The absorbance values were normalized to the weight of the scaffolds and expressed as absorbance/g. Constructs were assayed in triplicate.

Abbasi-Ravasjani S., Seddiqi H., Moghaddaszadeh A., Ghiasvand M.E., Jin J., Oliaei E., Bacabac R.G, & Klein-Nulend J. (2022). Sulfated carboxymethyl cellulose and carboxymethyl κ-carrageenan immobilization on 3D-printed poly-ε-caprolactone scaffolds differentially promote pre-osteoblast proliferation and osteogenic activity. Frontiers in Bioengineering and Biotechnology, 10, 957263.

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Viral Replication Assay in HeLa ACE2 Cells

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For the assessment of viral replication, 10,000 HeLa^ACE2 cells were seeded in each well of a 96-well plate (TPP Techno Plastic Products AG) and incubated for 48 h. Cells were infected with 50 µL of a mock-infection sample or purified virus (1000 TCID₅₀/mL) in an infection medium (5% DMEM) for 2 h at 37 °C. Supernatants were discarded and cells were washed twice with 100 µL of 1× PBS before being overlaid with a 100 µL of fresh infection medium. Plates were incubated for 48 h at 37 °C, and virus-containing supernatants were collected and subjected to viral RNA isolation.

Synowiec A., Jedrysik M., Branicki W., Klajmon A., Lei J., Owczarek K., Suo C., Szczepanski A., Wang J., Zhang P., Labaj P.P, & Pyrc K. (2021). Identification of Cellular Factors Required for SARS-CoV-2 Replication. Cells, 10(11), 3159.

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Determining Antioxidant Capacity via ORAC Assay

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Oxygen radical absorbance capacity (ORAC) assay was performed in a 96-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland), using fluorescein (Sigma-Aldrich, Merck) as a fluorescent probe, according to the procedures of Dávalos et al. (28 (link)). The measurements were performed using a microplate reader (FLUOstar OPTIMA; BMG Labtech, Offenburg, Germany) with fluorescence filters at 485 nm excitation and 520 nm emission. The reaction, performed at 37 °C, was started by thermal decomposition of 2,2’-azobis(2-methylpropionamidine) (AAPH; Sigma-Aldrich, Merck, Steinheim, Germany) in a 75-mM phosphate buffer (pH=7.4). ORAC values were calculated using the difference between the area under the fluorescein decay curve of the sample and the blank (net area under the curve). Standard curves were constructed using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich, Merck) at concentrations between 30 and 1500 μmol/mL. Regression equations between the net area under the curve and antioxidant concentration were calculated. Final values are expressed in μmol Trolox equivalents (TE) per mL of dry matter and analyses were performed in triplicate with NLC_BO and NLC_BO24h.

de Castro Reis L.V., Leão K.M., Speranza P., Ribeiro A.P., Macedo G.A, & Macedo J.A. (2020). Evaluation of Nanostructured Lipid Carriers Produced with Interesterified Buriti Oil. Food Technology and Biotechnology, 58(3), 284-295.

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Proliferation Assay of PBMC and T-Cells

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PBMC or enriched T-cells were suspended in RPMI tissue culture medium with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml) and 5% pooled and heat-inactivated human AB serum (complete medium). In total, 0.15×10⁶ cells in 100 µl complete medium were added to each well of tissue culture plates (96-well plate, Techno Plastic Products AG, TPP, Trasadingen Switzerland). Before the cells were added to the 96-well plate, 50 µl of reactants, medium, 400 setae, setae extract (100 ng/ml) or chitin (5 µg/ml) were added to the wells. Each reactant was added to the 96-well plate in sextuplicates. The plates were incubated in a humid atmosphere with 5% CO₂ at 37°C for 6 days in total. Eighteen hours before harvesting the cells, l µCi ³H-thymidine (20 Ci/mmol, GE Healthcare) was added to each well. The cell pellets were harvested and washed in a Harvester 96-Automated TOMTEC and the radioactivity was counted in a Wallac 1450 Beta scintillation counter (Perkin Elmer). The mean counts/min (ct/min) of six wells was calculated.
In parallel, cells incubated with setae, setae extract or medium only with no addition of ³H-thymidine were stained for CD3, CD25, and CD 19 after 6 days.

Holm G., Andersson M., Ekberg M., Fagrell B., Sjöberg J., Bottai M, & Björkholm M. (2014). Setae from Larvae of the Northern Processionary Moth (Thaumetopoea pinivora, TP) Stimulate Proliferation of Human Blood Lymphocytes In Vitro. PLoS ONE, 9(12), e113977.

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CAL 27 Cell Culture and Treatment

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Cell line, cell culture. The CAL 27 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 µg/mL) in a humidified atmosphere of 5% CO₂ at 37 °C, in a Binder incubator, until confluence. After 24 h, the cells were dissociated with trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA), counted using a Cellometer Mini Automated Cell Counter (Nexcelom Bioscience), and the cell viability was assessed by the trypan blue dye exclusion method. Then, the cells were cultured in 96-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) with a density of 8 × 10³ cells/well, being incubated under the same temperature and humidity conditions in the binder incubator.
After 24 h, which is necessary for monolayer formation, the cells of the different experimental variants were treated, for 24 and 48 h respectively with UBE, which was dissolved in DMSO 0.2% for obtaining six final concentrations. The negative control was performed using growth medium alone, and the impact of the solvent was tested.

Popovici V., Bucur L.A., Schröder V., Gherghel D., Mihai C.T., Caraiane A., Badea F.C., Vochița G, & Badea V. (2020). Evaluation of the Cytotoxic Activity of the Usnea barbata (L.) F. H. Wigg Dry Extract. Molecules, 25(8), 1865.

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Chronic TGF-β Inhibition Assay in Py2T Cells

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For the chronic inhibition experiment, Py2T cells were seeded in 96-well plates (Techno Plastic Products AG) with a seeding density of 1,800 cells per well in 80 μl of growth cell media. Only the 60 inner wells were used for analysis. To acquire sufficient sample size, five 96-well plates were used for single condition. After seeding, cells were allowed to recover for 36 h to reach 50% confluence. Cells were treated simultaneously with TGF-β₁ or vehicle (PBS) and small-molecule inhibitor or vehicle (DMSO) for 5 d, and medium was changed daily. All pipetting procedures were performed at room temperature using a Biomek FX Laboratory Automation Workstation (Beckman Coulter) supplied with 96-well pipetting pod.
In addition to experimental conditions treated with small-molecule inhibitors, at least five ‘uninhibited’ control conditions and five ‘untreated’ control conditions were included on each 96-well plate. Uninhibited control conditions were those in which TGF-β was applied to induce EMT in absence of any inhibitor. Untreated control conditions were those in which neither TGF-β nor inhibitor was applied and no EMT was induced.

Chen W.S., Zivanovic N., van Dijk D., Wolf G., Bodenmiller B, & Krishnaswamy S. (2020). Uncovering axes of variation among single-cell cancer specimens. Nature methods, 17(3), 302-310.

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