The tumoursphere assay was performed in accordance with previously reported protocols48 (link). Cells were collected by trypsinization and resuspended as single cells in cancer stem cell media containing serum-free DMEM/F12 (Invitrogen) supplemented with 100 units/mL B27 (Gibco), 10 µg/mL Insulin (Sigma), 20 ng/mL EGF (Sapphire Bioscience), and 20 ng/mL bFGF (Sapphire Bioscience). The cells were plated in a round-bottom 96-well ultra-low attachment plates (Corning) at densities of 1, 5, 20, and 50 cells/well. Sphere formation was recorded 5–7 days after incubation at 37 °C in a humidified atmosphere with 5% CO2. Cancer stem cell frequency was calculated using the limiting dilution software package (ELDA) on the website of Walter and Eliza Hall Institute of Medical Research (http://bioinf.wehi.edu.au/software/elda/index.html )49 . Only spheres with a size larger than 50 µm in diameter were counted. For ex vivo xenograft assay, cells were injected subcutaneously into the left flanks of female NOD/SCID mice at three cell densities (5 × 104, 1 × 104, and 1 × 104) in serum-free DMEM with Matrigel at 1:1 ratio. Tumours were detected by palpation.
Ultra low attachment 96 well round bottom plates
Manufactured by Corning
Sourced in United States
Ultra-low attachment 96-well round-bottom plates are laboratory equipment designed for cell culture applications. These plates feature a specialized surface treatment that minimizes cell attachment, promoting the formation of three-dimensional cell aggregates or spheroids. The round-bottom well design supports the growth and maintenance of these cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Epidermal growth factor (egf), by Sapphire Bioscience (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
35 mm glass bottom dish, by Cellvis (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Ix51 microscope, by Olympus (1 mentions)
Celigo cytometer, by Revvity (1 mentions)
Observer d1 microscope, by Zeiss (1 mentions)
Axio vison le software, by Zeiss (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Matrigel, by Corning (1 mentions)
Dp71 camera, by Olympus (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Powershot s50 camera, by Canon (1 mentions)
Aprotinin, by Merck Group (1 mentions)
11 protocols using ultra low attachment 96 well round bottom plates
1
Tumoursphere Assay and Xenograft
2
Establishment and Culture of CM Cell Lines
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The CM cell lines CRMM1, CRMM2, and CM2005.1 were originally established from recurrence patients [34 (link),35 (link)] and provided by the Liverpool Ocular Oncology Research Group. The authentication of the cell lines was performed using genotyping (Cellosaurus STR similarity search tool [36 (link)]) and negatively tested for mycoplasma infection using real time PCR [37 ]. Cells were cultured in complete medium containing F-12K medium with 10% FCS at 37 °C in a fully humidified atmosphere with 5% (v/v) CO2. Spheroid generation was performed as described in previous publications [28 (link),38 (link)]. Briefly, spheroids were generated by seeding 5 × 103 cells in round bottom 96-well ultralow attachment plates (Corning, Corning, NY, USA) containing 200 µL of complete medium.
3
Spheroid Generation in 96-well Plates
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The spheroids were generated by seeding 5 × 103 living cells in round bottom 96-well ultra-low attachment plates (Corning, Corning, NY, USA) in 100 µL of the respective cell culture medium. The medium was refreshed two times per week. The spheroid cultures were incubated in a humified incubator (37 °C, 5% CO2) for the indicated time of period. Compact spheroids could be generated on day 4 of the culture.
4
3D Invasion Assay using Spheroids
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Spheroids were formed in ultra-low attachment 96-well round bottom plates (Corning) and embedded in either 2.0 mg/mL Matrigel (BD Biosciences) or 3.0 mg/mL rat tail Type I Collagen (BD Biosciences) in a 35 mm glass bottom dish (Cellvis) and allowed to invade for up to 72 h incubated at 37 °C. Images were taken using an Olympus IX51 microscope 10× (0.30 NA air) with an Infinity2 CCD camera. For drug treatments, compounds were added directly to the Matrigel or collagen during the embedding process, as well as to the growth media added on top of the matrix.
Invasive area was quantified by measuring both the total spheroid area around the outer perimeter and the inner spheroid core in ImageJ/Fiji and taking the difference between the two measures. Spheroid circularity was utilized as an indirect measure of the chain-like invasion pattern and was quantified in ImageJ/Fiji by measuring the spheroid outer invasive perimeter.
Invasive area was quantified by measuring both the total spheroid area around the outer perimeter and the inner spheroid core in ImageJ/Fiji and taking the difference between the two measures. Spheroid circularity was utilized as an indirect measure of the chain-like invasion pattern and was quantified in ImageJ/Fiji by measuring the spheroid outer invasive perimeter.
5
Tumor Spheroid Formation and Analysis
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Complete growth medium containing 1 × 104 SK-Hep-1 cells (200 µL) was seeded into ultralow attachment 96-well round-bottom plates (Corning, USA) for 3 days, as described previously [25 –27 (link)]. Images were acquired when tumor spheroids were formed (Day 0). Then, 100 µL of plating medium was removed and replaced with the same volume of Matrigel. After Matrigel solidification, 100 µL of complete growth medium was added to each well. After 3 days, images were photographed and analysed using the Celigo™ cytometer (Nexcelom Bioscience, Lawrence, MA, USA).
6
Nanoparticle-Mediated Spheroid Modulation
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Cell lines were seeded on ultra-low attachment 96-well round-bottom plates (Corning; Corning, NY, USA) at 5000 cells per well. After 24 h, the formed spheroids were treated with empty nanoparticles or nanoparticles loaded with DOX, or DOX and miR-7-5p. Spheroids were cultured with nanoparticles for 6 days and pictures were captured using Observer D1 microscope (Zeiss; Oberkochen, Germany; 10× lens) equipped with Axio Vison LE software (Zeiss; Oberkochen, Germany). The area of spheroids was calculated using ImageJ tool (NIH; Bethesda, MD, USA). The results are presented as the percentage of spheroid area compared to controls.
7
Telomerase Inhibition in Progenitor and Stem Cells
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An equal number of cells from PCs and SDCs were seeded in 200 μl DMEM with 10% FBS and in 200 μl serum-free medium using 96-well culture plates and ultra-low-attachment 96-well round bottom plates (all, Corning, Costar, USA), respectively, in triplicate. EGF and bFGF were added every other day for the forming of SDCs. MST-312 (EMD, Millipore, USA) is a synthetic compound that has been shown to be a potent telomerase inhibitor (28 (link)). MST-312 powder was reconstituted to 5 mg/mL in dimethyl sulfoxide, and further diluted to the desired concentration. After growing the PCs and SDCs, MST-312 was added at concentrations of 1 and 10 μM and incubated for 24, 48, and 72 h (29 (link)).
8
Cell Culture Protocols for Cancer Research
9
Matrigel-based 3D Invasion Assay
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U251MG cells (1 × 103) were seeded into ultra-low attachment 96-well round-bottom plates (Corning) containing 200 µL of DMEM supplemented with 10% FBS and cultured for 2 days to form spheroids. Following spheroid formation, 100 µL of DMEM was replaced with 100 µL of 100% Matrigel (Corning) and incubated at 37 °C for 1 h before overlaying with 100 µL of DMEM containing 10% FBS. This was set as day 0. Micrographs of the spheroids were taken at ×10 magnification using an inverted Phase Contrast microscope (IX73, Olympus, Tokyo, Japan) coupled with a DP71 camera (Olympus). The area of cells invading into Matrigel was measured by ImageJ software using 8-bit images of spheroids. Briefly, the ‘find edges’ function with a minimum and a maximum threshold value of 0 and 50, respectively, was applied to each spheroid image. The tracing tool was then used to outline the spheroids. Area of spheroids measured on day 0 was subtracted from the area of spheroids measured on day 2 to obtain the area of cells invading into Matrigel. The data are presented relative to the shControl cells.
10
Hybrid Tumor Spheroid Formation
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