Deep well plate

Manufactured by Corning

Sourced in United States

The Deep Well Plate is a laboratory equipment designed to hold and process multiple liquid samples simultaneously. It features a grid of deep wells, providing increased volume capacity compared to standard well plates. The Deep Well Plate is a versatile tool used for various applications in life science research and sample preparation.

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Lab products found in correlation

Culture insert, by Corning (1 mentions) Human thrombin, by Merck Group (1 mentions) Falcon cell culture insert, by Corning (1 mentions) Cacl2, by Merck Group (1 mentions)

Close spelling variants of this product

6-deep well plates 24 deep‐well plates 96-deep-well plates 96-well deep well plate

4 protocols using deep well plate

Dialysis Culture Membrane Design

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A dialysis culture plate was prepared with a deep well plate (Corning, USA) and a culture insert (Corning, USA), which had a dialysis membrane (Spectrum, USA). The polycarbonate membrane of the culture insert was removed and replaced with a dialysis membrane, which had a molecular weight cutoff (MWCO) of 3 kDa and a surface area of 0.53 cm² (herein after, this is called “dialysis cup”). The bottom of the dialysis membrane was covered with a polyethylene film (Diversified Biotech, USA) to avoid mass transfer as the comparison to dialysis culture (hereinafter, this is called “blocked dialysis cup”) (Fig. 1a).Fig. 1

The culture condition in this study. (a) Schematic images of culture design. (b) Schedule of cytekines addition for the differentiation.

Fig. 1

Shinohara M., Choi H., Ibuki M., Yabe S.G., Okochi H., Miyajima A, & Sakai Y. (2019). Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture. Regenerative Therapy, 12, 14-19.

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Dialysis Culture Plate Protocol

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The dialysis culture plate was prepared in the same way as the previous study [17 (link)]. Briefly, the original membrane of a 6-well culture insert (Corning, New York, NY, USA) was replaced with a regenerated cellulose dialysis membrane (Spectrum Labs, San Francisco, CA, USA), which had a 3 kDa molecular weight cut off (MCWO). The dialysis membrane combined insert cup was inserted in the deep well plate (Corning,) (Figure 1a). During the differentiation, 2.5 mL of differentiation medium was added to the culture insert and 14.5 mL of dialysis medium was added in the deep well (Figure 1b). The molecule larger than 3 kDa remained in the culture insert during the culture on an orbital lab shaker. To compare the dialysis culture with a conventional suspension culture without dialysis, the dialysis membrane of the culture insert was sealed with polyethylene film (Diversified Biotech, Dedham, MA, USA) to block the mass transfer by the dialysis.

Choi H., Shinohara M., Ibuki M., Nishikawa M, & Sakai Y. (2021). Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System. Cells, 10(8), 2017.

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Quantifying AAV Titer via Flow Cytometry

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The procedure was described elsewhere and adapted for AAV transduction.⁷³ (link) HEK293SF-3F6 cells used for the transduction assay were infected with first-generation adenovirus (Ad ΔE1, ΔE3) at MOI 5 and seeded at 0.5 × 10⁶ cells/mL in suspension in 12-well plates (1 mL per well). Three dilutions of an AAV sample were prepared and 40 μL was added to the cells infected with adenovirus to ensure a range of GFP-positive cells of 2%–20% to ensure that cells were not transduced with more than one infectious viral particle. Positive control was an AAV sample with known titer and negative control was HEK293SF cells infected only with Ad (ΔE1, ΔE3). Infected cells were incubated for 24 h, transferred to deep-well plates (Corning) and pelleted by centrifugation for 2 min at 400 × g. Supernatant was removed and cells were resuspended in PBS with 2% formaldehyde. Fixed cells were read on a flow cytometer and percentage of GFP-positive cells was used to calculate the infectious viral particles titer (IVP/mL). The following formula was used to calculate titer:

I V P / m L = \frac{% G F P s a m p l e - % G F P n e g c t l}{100} * D i l u t i o n F a c t o r * \frac{A v e r a g e C e l l C o u n t (\frac{c e l l s}{m L})}{40 μ L} * 1000 μ L / m L .

Jalšić L., Lytvyn V., Elahi S.M., Hrapovic S., Nassoury N., Chahal P.S., Gaillet B, & Gilbert R. (2023). Inducible HEK293 AAV packaging cell lines expressing Rep proteins. Molecular Therapy. Methods & Clinical Development, 30, 259-275.

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In Vitro Organotypic Skin Model

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To establish the in vitro OTCs, human dermal fibroblasts (1 × 10⁵) were resuspended in a fibrin matrix derived from pig plasma cryoprecipitate diluted in growth medium, supplemented with 800 μg/mL Amchafibrin (Fides Ecopharma, Barcelona, Spain) and 2.5 U of human thrombin diluted in 0.025 mM CaCl₂ (Sigma-Aldrich). The mixture was placed in a six-well transwell plate with 1-μm pore polyester membrane (Falcon cell culture insert, Corning Life Sciences, NY, USA). After gelation (2 h at 37°C), human keratinocytes (1 × 10⁶ cells/well) were seeded onto the gel. When keratinocytes reached confluence, the insert was transferred to deep-well plates (Corning Life Sciences) and cultured at the air-liquid interface for 2 weeks to allow proper epithelium differentiation. To this end, the growth medium was removed from the surface of the culture and differentiation medium (2:1 of DMEM/Ham’S F-12 mixture supplemented with 0.5% FBS, 5 μg/mL insulin, 8 ng/mL cholera toxin, 0.4 μg/mL hydrocortisone, 2.4 ng/mL adenine, 1.3 ng/mL triiodothyronine, 1% penicillin/streptomycin, 1.5 ng/mL EGF, and 50 μg/mL ascorbic acid) was added under the insert. The differentiation medium was renewed twice a week for 2 weeks until OTC harvest. In order to confirm tissue stability, some OTCs remained in culture up to 4 weeks.

Gálvez V., Chacón-Solano E., Bonafont J., Mencía Á., Di W.L., Murillas R., Llames S., Vicente A., Del Rio M., Carretero M, & Larcher F. (2020). Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome. Molecular Therapy. Methods & Clinical Development, 18, 280-290.

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