Black bottomed 96 well plate

Manufactured by Corning

The Black-bottomed 96-well plate is a laboratory equipment designed for use in various scientific experiments and assays. The plate features 96 individual wells with a black-colored bottom, which can help to reduce background signal and improve optical performance in certain applications. The core function of this product is to provide a standardized platform for the containment and manipulation of small liquid samples during experimental procedures.

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Lab products found in correlation

Phenol red free dmem, by Thermo Fisher Scientific (2 mentions) Fd10s, by Merck Group (2 mentions) Varioskan lux, by Thermo Fisher Scientific (2 mentions) Fluostar optima, by BMG Labtech (1 mentions) Fd250s, by Merck Group (1 mentions)

Spelling variants of this product

96-well plates (3030 citations) 96 wells (10 citations) Clear-bottomed 96-well black plates (3 citations) Black-walled optically clear-bottomed 96-well plates (2 citations) Black clear‐bottomed Corning Costar 96‐well polystyrene plates (2 citations)

4 protocols using black bottomed 96 well plate

Transepithelial Permeability Assay

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After the TER measurement, the basal medium was replaced with phenol red-free DMEM (#21063-029; Thermo Fisher Scientific) supplemented with 5% FBS. The apical medium was replaced with medium containing 1 mg/ml of FITC-dextran with a molecular mass of 10 kD (FD10S; Sigma-Aldrich). After incubation at 37°C for 2 h, the fluorescence intensity of the basal medium was measured using a microplate reader (VARIOSKAN LUX; Thermo Fisher Scientific) in a black-bottomed 96-well plate (Corning). The apparent permeability coefficient (P_app) was calculated using the following equation (Watson et al., 2001 (link); Van Itallie et al., 2008 (link)):

P_{a p p} [c m / s] = \frac{d Q}{d t} ∙ \frac{1}{A ∙ C},

where dQ [mg] is the amount of tracer transported to the basal acceptor compartment during incubation time dt [s], A [cm²] is the area of the filter and C [mg/cm³] is the initial concentration of the tracer in the apical donor compartment.

Higashi T., Saito A.C., Fukazawa Y., Furuse M., Higashi A.Y., Ono M, & Chiba H. (2022). EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier. The Journal of Cell Biology, 222(1), e202204079.

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Extracellular ROS Evaluation in BEAS-2B Cells

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BEAS-2B cells were seeded into 24 well plates at a density of approximately 1.5×10⁵ cells/ml. After 24 h, the cells were treated with nanoclays and byproducts dispersed in media through use of a bath sonicator at their respective, determined IC₅₀ value; cells exposed to only media served as control samples. After 24, 48, and 72 h of treatment, 50 μl of the media was transferred from the 24 well plate to its respective well in a black-bottomed 96 well plate (Corning, Inc.). Subsequently, 50 μl of PBS was added to each well in the 96 well plate. Fifty μl of the extracellular reactive oxygen species (ROS) assay reagent, Lumigen ECL Plus (Lumigen, Inc.), was also added to each well. The samples were subsequently incubated at room temperature for 5 min, in the dark before luminescence was evaluated using a FLUOstar OPTIMA plate reader (BMG LABTECH) at 600 nm. Media and treated media containing nanoclays or byproducts suspended in solution served as blanks. Respective cellular measurements of the samples were evaluated after subtracting the blanks in order to determine the effect treatment had on extracellular ROS. It has been determined that Lumigen reagent assays generate chemiluminescent responses specific to extracellular ROS.³⁷ Four replicates were performed for each treatment.

Wagner A., White A.P., Stueckle T.A., Banerjee D., Sierros K.A., Rojanasakul Y., Agarwal S., Gupta R.K, & Dinu C.Z. (2017). Early assessment and correlations of nanoclay’s toxicity to their physical and chemical properties. ACS applied materials & interfaces, 9(37), 32323-32335.

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Measuring Epithelial Barrier Permeability

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After TER measurement, the basal medium was replaced with phenol red-free DMEM (Life Technologies) supplemented with 10% FBS. The apical medium was replaced by the medium with 1 mg/ml FITC-dextran of molecular mass of 3–5 kDa (FD4; Sigma-Aldrich), 10 kDa (FD10S; Sigma-Aldrich), or 250 kDa (FD250S; Sigma-Aldrich). The cells were incubated in a 5% CO₂ incubator at 37°C for 2 h, and the basal medium was collected. Fluorescence intensity of the medium was measured by microplate reader equipment (VARIOSKAN LUX; Thermo Fisher Scientific) in a black-bottomed 96-well plate (Corning). Standard curves were determined by measuring the fluorescence intensities of a serial dilution series of the FITC-dextran. Blank measurement of the medium without FITC-dextran was subtracted from the sample values. The apparent permeability coefficient (P_app) was calculated using the following equation:

where dQ is the amount of tracer transported to the acceptor (basal) compartment during the time period dt, A is the area of the filter, and C is the initial concentration of the donor (apical) compartment.

Saito A.C., Higashi T., Fukazawa Y., Otani T., Tauchi M., Higashi A.Y., Furuse M, & Chiba H. (2021). Occludin and tricellulin facilitate formation of anastomosing tight-junction strand network to improve barrier function. Molecular Biology of the Cell, 32(8), 722-738.

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Extracellular ROS Measurement in BEAS-2B Cells

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BEAS-2B cells were seeded in a 12 well plate at a density of 1.5 × 10⁵ cells/ml. After 24 h, the cells were exposed to 100 or 300 µg/ml of PLACC900 dispersed in media as previously described. After 24, 48, and 72 h of exposure, 50 µl of media from each treatment was transferred to a black-bottomed 96 well plate (Corning, Inc.). Subsequently, 50 µl of PBS and 50 µl of Lumigen ECL Plus (Lumigen, Inc.) were added to each well, and the samples were incubated for 5 min in the dark. Luminescence was read at 600 nm via the FLUOstar OPTIMA plate reader. Media as well as PLACC900 dispersed in media, at each dose, served as blanks. Extracellular reactive oxygen species (ROS) was calculated by subtracting PLACC900 luminescence (determined via subtraction of media from the PLACC900 + media blanks) from the respective cellular measurements.

Wagner A., White A.P., Tang M.C., Agarwal S., Stueckle T.A., Rojanasakul Y., Gupta R.K, & Dinu C.Z. (2018). Incineration of Nanoclay Composites Leads to Byproducts with Reduced Cellular Reactivity. Scientific Reports, 8, 10709.

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