Ultra low attachment surface

Manufactured by Corning

Sourced in United States

The Ultra-low attachment surface is a specialized laboratory equipment designed to create a cell culture environment that minimizes cell attachment. It features a highly hydrophilic surface that prevents the adhesion of cells, allowing them to grow in a suspension culture. This product is intended to maintain the undifferentiated state of cells and promote the formation of spheroids or aggregates, which can be useful for various cell culture applications.

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51 protocols using ultra low attachment surface

Suspension Culture and Anoikis Assay

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Cell survival under suspension culture condition was determined. Briefly, cells at a density of 5 × 10³ cells/well with antibodies were seeded into 96-well plates with an Ultra-Low Attachment Surface (Corning, NY, USA) and incubated for 7 days in the absence of serum. Cell viability was determined using the colorimetric WST-1 assay as described above.
Cells (5 × 10⁵) were seeded in the presence of antibodies into six-well plates with an Ultra-Low Attachment Surface (Corning) to induce anoikis. Cells were washed and stained with 5 µl of annexin V and 5 µl of PI per 1 × 10⁵ cells for 15 min at r.t. in the dark, and the percentage of apoptotic cells was analyzed using flow cytometry. The cells were harvested after the induction of anoikis, washed with PBS, and lysed for immunoblot analysis.

Ahn H.M., Ryu J., Song J.M., Lee Y., Kim H.J., Ko D., Choi I., Kim S.J., Lee J.W, & Kim S. (2017). Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity. Theranostics, 7(3), 594-613.

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Tumor Sphere Formation and Stem Cell Frequency Assay

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Cells from the indicated groups were seeded at a density of 1000 cells per well in a 6-well plate with an ultra-low attachment surface (Corning). Tumor spheres were generated in serum-free DMEM/F-12 and supplemented with 20 ng/mL EGF, 20 ng/mL FGF-basic, 1% N-2, and 2% B-27. After 10 days, photomicrographs of tumor spheres were taken, and the size and number of tumor spheres (with a diameter > 50 μm) were measured by an assessor in a blinded manner. The ratio of sphere formation was calculated as the number of spheres per well divided by the number of seed cells per well. The spheres were dissociated into single cells with trypsin–EDTA for cell passage and subsequent analysis.
For the in vitro extreme limiting dilution assay, single-cell suspensions of the indicated groups were serially diluted to a decreasing cell density (160, 80, 40, 20, 10, 5, and 1 cell per well) and seeded into 96-well plates with an ultra-low attachment surface (Corning). The cells were then incubated under sphere formation conditions (as described above) for 10 days. The presence of spheres in each well was recorded, and the stem cell frequency was calculated using the ELDA tool (https://bioinf.wehi.edu.au/software/elda) [29 (link)].

Shi H., Tan Z., Duan B., Guo C., Li C., Luan T., Li N., Huang Y., Chen S., Gao J., Feng W., Xu H., Wang J., Fu S, & Wang H. (2024). LASS2 enhances chemosensitivity to cisplatin by inhibiting PP2A-mediated β-catenin dephosphorylation in a subset of stem-like bladder cancer cells. BMC Medicine, 22, 19.

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Sphere-forming Assay for Cancer Cells

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For sphere-forming assay, we used ReproFF2 medium (Repro cell, Kanagawa, Japan), which is a new feeder-free culture medium for human ES/iPS cells. We also used the Ultra-low attachment surface (Corning, Corning, NY, USA), which is coated with a hydrogel layer and prevents attachment of cancer cells growing in an anchorage-dependent manner.^{40 (link)} AGS and HepG2 cells were cultured in serum-free ReproFF2 medium containing 5 ng/ml bFGF with 5 μM DZNep or 1 μM SAHA in a 96-well plate with an Ultra-low attachment surface. The AGS and HepG2 cells were plated at 200 and 500 per well, respectively. Fresh culture medium was added on day 5 of the culture period. The number of spheroids was counted 10 days after drug treatment. Experiments were carried out in triplicate.

Hibino S., Saito Y., Muramatsu T., Otani A., Kasai Y., Kimura M, & Saito H. (2014). Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells. Oncogenesis, 3(5), e104-.

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Cell Survival and Anoikis Assays

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To determine cell survival under suspension culture conditions, cells (1.5 × 10⁴) were seeded into 96-well plates with an Ultra-Low Attachment Surface (Corning, NY), and then incubated for up to 7 days in the absence of serum. Cell viability was determined using the colorimetric WST assay as described above. To induce anoikis, cells (5 × 10⁵) were seeded into 6-well plates with an Ultra-Low Attachment Surface (Corning) for 48 or 72 h. Cells were then stained with 5 μl of annexin V and 5 μl of propidium iodide (PI) per 1 × 10⁵ cells for 15 min at 25 °C in the dark, and the percentage of apoptotic cells was analyzed by flow cytometry.

Lee Y., Yoon J., Ko D., Yu M., Lee S, & Kim S. (2021). TMPRSS4 promotes cancer stem–like properties in prostate cancer cells through upregulation of SOX2 by SLUG and TWIST1. Journal of Experimental & Clinical Cancer Research : CR, 40, 372.

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3D Cell Culture Assay for Colony Formation

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Cells were plated in 24-well plate with Ultra-Low Attachment surface (Corning Inc., Corning, NY, USA) at a density of 2.5 × 10³ cells per well and cultured for 21 days in RPMI1640 with 10% FBS and 0.3% agarose. RPMI1640 with 10% FBS were added to the top of agar layer and exchanged every 3 days. Nine images per well were taken every day using IncuCyteZOOM. The colony formation was defined as the cell aggregates occupying an area at least 8000 μm² (about 100 μm diameter) and the occupied area were measured using IncuCyte ZOOM 2015A software.24 (link)

Hashida S., Yamamoto H., Shien K., Miyoshi Y., Ohtsuka T., Suzawa K., Watanabe M., Maki Y., Soh J., Asano H., Tsukuda K., Miyoshi S, & Toyooka S. (2015). Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib. Cancer Science, 106(10), 1377-1384.

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Culturing Cells in Serum-Free Media

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When cell confluence reached 80%, the cells were detached using 1×trypsin-EDTA. Cells free from serum were suspended individually in DMEM/F12 medium supplemented with 1% b27 supplement, 1% antibiotic-antimycotic (Invitrogen), 20 ng/mL epidermal growth factor (EFG; Invitrogen, Grand Island, NY, USA), and 20 ng/mL basic fibroblast growth factor (bFGF; Invitrogen, Seoul, Korea) [DMEG(+)growth factor (GF)], DMEM-high glucose with FBS {DMEM(+)FBS, or DMEM-high glucose without FBS [DMEM(-)FBS]}. Cells were subsequently cultured in ultra-low attachment 24-well plates (24 well plate coated with Ultra-Low Attachment Surface, Corning, NY, USA) at a density of 10000 cells per well, and were incubated at 37℃ with 5% CO₂.

Choi S.H., Lee S.W., Ok M., Kim K.S., Kim S, & Ahn S.H. (2017). Gene Expression Profiling of Hepatocellular Carcinoma Derived Cancer Stem Like Cell under Hypoxia. Yonsei Medical Journal, 58(5), 925-933.

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Sphere Formation Assay for Cell Stemness

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Sphere formation assays were performed according to the above-described procedures following previously reported protocols [29 (link)]. Briefly, 500 cells were seeded in 6-well plates coated with Ultra-Low Attachment Surface (Corning, USA), followed by culturing for 3 weeks in DMEM/F12 medium (Gibco, USA) supplemented with B27 (1:50;Gibco, USA), 20 ng/ml EGF (Life Technologies, USA) and 20 ng/ml basic FGF (Life Technologies, USA). The spheres were observed, imaged and counted under a microscope.

Lei J.J., Peng R.J., Kuang B.H., Yuan Z.Y., Qin T., Liu W.S., Guo Y.M., Han H.Q., Lian Y.F., Deng C.C., Zhang H.J., Chen L.Z., Feng Q.S., Xu M., Feng L., Bei J.X, & Zeng Y.X. (2015). NOP14 suppresses breast cancer progression by inhibiting NRIP1/Wnt/β-catenin pathway. Oncotarget, 6(28), 25701-25714.

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Mammosphere Formation Assay

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Single-cell suspension was suspended in sphere-culturing medium (Stemcell Technologies) at a density of 1000 cells/well and seeded into 24-well plates with ultra-low attachment surface (Corning) at 37 °C in a humidified atmosphere containing 5% CO₂. For evaluation, the mammospheres were counted and photographed under an inverted microscope (Olympus) after 2 weeks.

Jiang H., Zhou C., Zhang Z., Wang Q., Wei H., Shi W., Li J., Wang Z., Ou Y., Wang W., Wang H., Zhang Q., Sun W., Sun P, & Yang S. (2020). Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1. Nature Communications, 11, 5129.

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Cell Survival Under Suspension Culture

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Cell survival under suspension culture conditions was determined. Briefly, cells were seeded into 96-well plates with an Ultra-Low Attachment Surface (Corning, NY, USA) at a density of 3 × 10⁴ cells/well and incubated for 3 or 5 days in the absence of serum. Cell viability was determined using the colorimetric WST assay as described above.
Cells (3 × 10⁵) were seeded into 6-well plates with an Ultra-Low Attachment Surface for 48 h to induce anoikis. Cells were washed and stained with annexin V and propidium iodide (PI) for 15 min at room temperature in the dark. The percentage of apoptotic cells was analyzed using flow cytometry.

Ko D, & Kim S. (2017). Cooperation between ZEB2 and Sp1 promotes cancer cell survival and angiogenesis during metastasis through induction of survivin and VEGF. Oncotarget, 9(1), 726-742.

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Anoikis Induction and Apoptosis Analysis

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Cells (5 × 10⁵) were seeded in the absence or presence of compound into 6-well plates with an Ultra-Low Attachment Surface (Corning, NY, USA) for 48 h to induce anoikis, as described previously^{25 (link)}. Cells were washed and stained with 5 μl of annexin V and 5 μl of propidium iodide (PI) per 1 × 10⁵ cells for 15 min at room temperature in the dark, and the percentage of apoptotic cells was analyzed using flow cytometry. The cells were harvested after the induction of anoikis, washed with PBS, and lysed for immunoblot analysis. Where indicated, annexin V or PI-positive cells relative to the total cells were counted to determine the percentage of apoptotic cells.

Kim S., Ko D., Lee Y., Jang S., Lee Y., Lee I.Y, & Kim S. (2019). Anti-cancer activity of the novel 2-hydroxydiarylamide derivatives IMD-0354 and KRT1853 through suppression of cancer cell invasion, proliferation, and survival mediated by TMPRSS4. Scientific Reports, 9, 10003.

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