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Non adherent 96 well plate

Manufactured by Corning
Sourced in United States

The Non-adherent 96-well plate is a laboratory equipment designed to prevent cell attachment to the plate surface. It features a special coating that inhibits cell adhesion, allowing for the cultivation of suspension cells or the formation of cellular spheroids. The plate provides a consistent and controlled environment for various cell-based applications, such as drug screening, cytotoxicity assays, and 3D cell culture experiments.

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6 protocols using non adherent 96 well plate

1

Limiting Dilution Assay for Breast Cancer Stem Cells

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For the limiting dilation assays (LDA), MDA-MB-231 and BT-549 primary tumour spheres (TS), obtained from 21-day old spheroids, were mechanically disaggregated and counted before being seeded in non-adherent 96-well plates (Corning). Next, serial dilutions were performed to achieve 200 cells/well down to 1 cell/well. Spheroid formation was monitored until day 21 by determining the number of TS per well.
For the secondary and tertiary TS formation assays, primary TS were mechanically dissociated after which 200.000 cells were cultured in ultralow attachment 100 mm plates (Falcon) in the presence of JQ1 (200 nM) for 3 days. Next, the number of secondary TS per plate were determined. Secondary TS were again dissociated and left in the presence of the drug until day 6, at which the number of tertiary TS per plate was determined. TS pictures were taken at the two time points using an inverted microscope (Nikon).
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2

Sphere Formation Assay for IL-7 Response

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To examine sphere forming capacity in response to IL-7, the sphere formation assay was performed as described previously69 . Cells were seeded on nonadherent 96-well plates (Corning) at 2 × 102/100 μL/well (n = 20) with or without IL-7 and maintained for one week. The medium was not changed or added so as to disturb the formation of sphere. Visible spheres (>100 μm in diameter) were counted under a microscope (×10) after 7-day incubation. For serial passages of the sphere, spheres were dissociated into single cells using enzymatic digestion with trypsin-EDTA (Life Technologies). The total number of spheres formed from 20 wells was counted each experiment, and the average for three independent experiments was calculated.
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3

Spheroid-Monolayer Co-Culture Assay

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The method was adapted from Iwancki et al (51 (link)). Glass bottom 8-chamber slides were coated with 0.1 μg/ml fibronectin. Primary FTECs were directly plated into each well (2×104 cells/well) at passage zero and grown to 100% confluency (72 hrs) before labeling with CellTracker Green (1:1000). In parallel, 5×103 TYK-nu cells were plated in a non-adherent 96-well plate (Corning) to form spheroids for 72 hrs. Spheroids were directly transferred from 96-well plate to the FTEC monolayer and incubated for 12 hrs before fixation and staining for actin (AlexaFluor 647 phalloidin, Thermo Scientific). Images were collected on a Zeiss LSM 510 Meta Confocal microscope and images processed with ImageJ.
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4

Anchorage-Independent Cell Culture Assay

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Cells grown in an anchorage-independent condition were trypsinized, washed twice with PBS, and resuspended in serum-free medium consisting of a 1:1 mixture of F-12 Nutrient Mixture (Ham's F-12) and DMEM, supplemented with 20 ng/mL epithelial growth factor (Invitrogen, Carlsbad, CA, USA), 20 ng/mL basic fibroblast growth factor (Millipore, Billerica, MA, USA), 0.5 nM hydrocortisone (Sigma-Aldrich), B-27 supplement (GIBCO), 2 mM L-glutamine (GIBCO), and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Cells were plated on a non-adherent 96-well plate (Corning, Corning, NY, USA) at 10, 100, or 1000 cells/100 µL/well. To inhibit TG2 activity, some cells were treated with 0.1, 0.5, and 1.0 mM cysteamine (Sigma-Aldrich). In some wells, 10 µg/mL anti-IL-6 mAb (eBioscience, San Diego, CA, USA) or 10 ng/mL recombinant IL-6 (eBioscience) was added. The medium was replaced every 3 days. Visible spheres were counted under a microscope on day 8 post-plating.
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5

3D Spheroid Formation and Growth Assay

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3D-Spheroid formation and growth assay was performed as described previously.56 (link),57 (link) Briefly, the detached cells were resuspended in GBO medium with a minor modification (without insulin) according to a previously published report.61 (link) Cells were seeded in the non-adherent 96-well plate (Corning, NY) at 400 cells/well. Half of the medium was changed every 2 days. The spheroid formation and growth was monitored by a microscope with a real-time camera (EVOS FL Auto Imaging System, Life Technologies, Carlsbad, CA, USA). Photographs of tumor spheres were taken at the indicated time points and the area of the spheroids was measured to reflect the growth status over time.
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6

Serum-Free Sphere Formation Assay

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Cells grown in an anchorage-independent condition were trypsinized, washed twice with PBS, and resuspended in serum-free medium consisting of a 1:1 mixture of F-12 Nutrient Mixture (Ham's F-12) and DMEM, supplemented with 20 ng/mL epithelial growth factor (Invitrogen, Carlsbad, CA), 20 ng/mL basic fibroblast growth factor (Millipore, Billerica, MA), 0.5 nM hydrocortisone (Sigma-Aldrich), B-27 supplement (GIBCO), 2 mM L-glutamine (GIBCO), and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin). Cells were plated on a non-adherent 96-well plate (Corning, Corning, NY) at 10, 100, or 1000 cells/100 µL/well. The medium was replaced every 3 days. Visible spheres were counted under a microscope on day 8 post-plating.
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