To test their ability for growth and sphere formation in suspension, the cells were trypsinized and passed through a 40-µm filter to obtain a single cell suspension. In total, 10 ml of media containing 2×104 cells was added to 96-well ultra-low attachment plates (Corning Inc., Corning, NY, USA). After 7 days, the plates were visually assayed for the formation of floating spheres. To assess the ability of primary spheres to form secondary spheres, the primary spheres were collected by centrifugation (1,000 × g for 5 min) and again digested to single-cell suspensions, and passed through a 40-µm filter. A total of 10 ml of medium containing 2×104 cells was added to 96-well ultra-low attachment plates (Corning Inc.). To determine the viability and self-renewal abilities of the constituent cells, after 7 days, secondary spheres were collected and placed into adherent plates to assess their colony-formation patterns.
Ultra low attachment 96 well plate
Manufactured by Corning
Sourced in United States, United Kingdom, Germany, Canada, China, France, Japan, Switzerland
Ultra-low attachment 96-well plates are a specialized laboratory equipment designed for cell culture applications. These plates feature a unique surface coating that minimizes cell attachment, promoting the formation of 3D cell aggregates or spheroids. The core function of these plates is to provide an environment that encourages the growth and development of cells in a controlled, non-adhesive manner.
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Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (41 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (40 mentions)
B27 supplement, by Thermo Fisher Scientific (31 mentions)
Dmem f12, by Thermo Fisher Scientific (24 mentions)
Heparin, by Merck Group (23 mentions)
Epidermal growth factor (egf), by Merck Group (22 mentions)
Methylcellulose, by Merck Group (18 mentions)
Matrigel, by Corning (15 mentions)
Hydrocortisone, by Merck Group (10 mentions)
24 well ultra low attachment plate, by Corning (10 mentions)
Insulin, by Merck Group (10 mentions)
N2 supplement, by Thermo Fisher Scientific (10 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Matrigel, by BD (9 mentions)
Axio observer z1, by Zeiss (9 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (9 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (8 mentions)
Stereomicroscope, by Olympus (8 mentions)
Heparin, by STEMCELL (7 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (7 mentions)
511 protocols using ultra low attachment 96 well plate
1
Sphere Formation and Self-Renewal Assay
2
MKN45 Spheroid Body Formation
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MKN45 cells were cultured in 1640 medium, supplemented with 10% FBS, and plated at the density of 1 × 106 cells per 75 cm2 flask. Then, we passaged them upon cell confluence. Spheroid bodies were derived by placing the parental cells into serum‐free 1640 culture medium (containing 1% N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 1% antibiotic mixture (100 mg/mL streptomycin and 100 U/mL penicillin G, Gibco), 20 ng/mL FGF2 (Chemicon) and 100 ng/mL EGF (Chemicon). The parental cells were plated in 96‐well ultra‐low attachment plate (Corning) at 100 cells per well. After two weeks, the number of suspension spheres was counted under the microscope (Olympus) and spheroid body formation was calculated. When the primary spheroid body reached the size of approximately 200‐500 cells per spheroid body, the spheroid bodies were separated at a density of 1000 cells per ml and 100 single‐cell suspension (100 μL) was seeded in each well of 96‐well ultra‐low attachment plate (Corning) in the serum‐free medium. After 2 weeks, the spheroid body formation of the second‐generation spheroid body was observed.
3
Tumorigenic Spheroid Formation Assay
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For tumorigenic spheroid formation, approximately 100 cells per well were seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell, MA, USA). The tumorigenic spheroids were photographed with the HoloMonitoring and confocal microscopy as live images. Cells were suspended in serum-free DMEM/F12 (1:1) culture medium supplemented with B27. Approximately 100–150 cells per well were seeded in an ultra-low attachment 96-well plate (Corning Inc., Lowell, MA, USA). The effect of carcinogenic regimen of 17β-estradiol (E2) was evaluated by E2 treatment (100 pg/mL) on the day of seeding cells. Spheroids were grown for 27 days in liquid culture. A total of 15 spheroids with a minimum diameter of 50 mm were counted in each experimental group. Data were analyzed by ANOVA; Tukey’s HSD test was used for multiple comparisons. Cells obtained from spheroids were analyzed by immunofluorescence, FACS, or immunoblotting, as described previously [7 ,8 (link)].
4
Tumor Spheroid Generation and Treatment
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Tumor spheroids were generated using 96-well ultra-low attachment (ULA) plates (Corning, NY, USA) as described previously [33 (link)]. During assay establishment, various cell numbers (1000–20 000) were seeded in 100 μl cell culture medium per well and centrifuged at 1000x g, for 10 min at room temperature. Spheroids were grown for 3 days before treatment with BH3 mimetics.
5
3D Spheroid Formation of HTR8/SVneo Cells
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96-well ultra-low attachment (ULA) plates (Corning) were used to promote the self-assembly of HTR8/SVneo cells into three-dimensional (3D) cellular spheroids. HTR8/SVneo cells were seeded at 1,000 cells/mL, 5,000 cells/mL and 10,000 cells/mL into the ultra-low attachment plates. Media was replaced every 48 hours post-seeding by aspirating 100 µL of existing media, being careful to not disturb the spheroid, and dispensing 100 µL of fresh media into each well.
6
Myometrial Spheroid Culture Protocol
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Myometrial spheroids were cultured as previously described [21 (link), 22 (link)]. Briefly, cells were plated in 96-well ultra-low attachment plates (Corning Costar), cultured in mesenchymal stem cell medium (Lonza, PT-3001), and incubated at 37 °C in a humidified incubator with 5% CO2 for at least 48 h. Spheroids were formed and evaluated under an inverted microscope.
7
Evaluating TNBC Tumorsphere Viability
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The TNBC cells were cultured in NanoCulture plates (2 × 104/well, SCIVAX Corporation, Kanagawa, Japan) and 96-well ultralow attachment plates (4 × 103/well, Costar®, New York, NY, USA). Cells transfected with 10 nM scramble or siRNA against DYRK1B were cultured for 7 d to observe tumorsphere of the control and silenced tumorsphere under microscope. Tumorsphere viability was evaluated by 3D CellTiter Glo (G9681, Promega) or Calcein AM (Green)/Ethidium homodimer-1 (EthD-1, Red) (LIVE/DEAD® Viability/Cytotoxicity Kit, ThermoFisher Scientific) to differentiate live and dead cells. Tumorsphere viability was evaluated and quantitated by Fluoroskan Ascent FL reader (Thermo Fisher Scientific), as previously described [17 (link)].
8
Spheroid Formation and Viability Analysis
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The protocols for forming spheroids and measuring cell viability by calculated spheroid volume were adapted from Vinci and coworkers 23 . Briefly, 1000 cells per well were seeded in 96‐well, ultralow attachment plates (Costar, Washington, DC) and incubated for 72 h. The spheroids were then treated with chemotherapy as described earlier. The spheroids were photographed using an IX81‐motorized inverted microscope (Olympus, Shinjuku, Tokyo, Japan) 96 h after treatment, and the software Cell^P (Olympus) was used to calculate the spheroid volume. The average radius of the spheroids was calculated, and spheroid volumes computed with the formula V = 4/3πr3, based on the assumption that the spheroids were approximately spherical.
9
Spheroid Formation Assay Protocol
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Spheroid formation assay were performed as previously described26 (link), cells were digested with trypsin, and cultured with 2% matrigel in 96-well ultralow attachment plates (Costar). Spheroid formation was initiated by centrifugation at 850 × g for 10 min and the spheroids were analyzed at the indicated time points. Spheroids were treated with ethanol (control) or doxycycline 48 h. At indicated time points, images were captured by a general optical microscope with a camera (Axiovert 100 M, Germany). Spheroid volume was calculated based on image analysis by area determination using image J software.
10
Spheroid Formation and Imaging Assay
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For spheroid formation, cells were trypsinized, counted, mixed with 2% matrigel in culture medium, and placed in 96-well ultralow attachment plates (Costar). Spheroid formation was initiated by centrifugation at 850 × g for 10 min, and cultures were incubated for the times indicated. Fresh growth medium, containing either ethanol or doxycycline (0.5 mg/ml), was administered every 48 hr. Spheroid size was determined at the times indicated by automated imaging on an inverted microscope (Axiovert 100M, Carl Zeiss).
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