PLC/PRF/5 cells were resuspended to a cell density of 5000 cells/ml, washed to remove serum and suspended in serum-free DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20 ng/ml human recombinant basic fibroblast growth factor (Merck KGaA), 20 ng/ml human recombinant epidermal growth factor (Merck KGaA), 2% B27 supplement without vitamin A (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml of streptomycin in ultra-low attachment 6-well plates (Corning Inc.). Fresh DMEM/F12 with 20 ng/ml human recombinant basic fibroblast growth factor, 20 ng/ml human recombinant epidermal growth factor, 2% B27 was added to the ultra-low attachment plates every two days. Four days later, the tumor spheres were collected by gentle centrifugation with 100 × g for 4 min at 37°C and digested by Accutase (Sigma-Aldrich; Merck KGaA) for 5 min at 37°C to form a single cell suspension for subsequent experiments. Tumor spheres were centrifuged with 100 × g for 3 min at 37°C to remove the enzyme and resuspended with 20 ng/ml human recombinant basic fibroblast growth factor, 20 ng/ml human recombinant epidermal growth factor and 2% B27 in ultra-low attachment 6-well plates (Corning Inc.) and allowed to reform spheres. The spheres were passaged every 4 days.
Ultra low attachment 6 well plate
Manufactured by Corning
Sourced in United States, China, France
The Ultra-low attachment 6-well plates are designed to create a controlled, non-adherent cell culture environment. The plates feature a hydrophilic, low-attachment surface that prevents cells from adhering to the plate, allowing for the formation of 3D cell spheroids or aggregates.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Thermo Fisher Scientific (98 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (79 mentions)
B27 supplement, by Thermo Fisher Scientific (64 mentions)
Dmem f12, by Thermo Fisher Scientific (48 mentions)
N2 supplement, by Thermo Fisher Scientific (33 mentions)
Epidermal growth factor (egf), by Merck Group (32 mentions)
Insulin, by Merck Group (30 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (23 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (22 mentions)
Heparin, by Merck Group (19 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (18 mentions)
Bovine serum albumin (bsa), by Merck Group (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Hydrocortisone, by Merck Group (11 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (11 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (11 mentions)
Insulin, by Thermo Fisher Scientific (11 mentions)
Basic fibroblast growth factor (bfgf), by BD (9 mentions)
Matrigel, by Corning (9 mentions)
Epidermal growth factor (egf), by R&D Systems (8 mentions)
553 protocols using ultra low attachment 6 well plate
1
Serum-Free Tumor Sphere Culture
2
Culturing Tumor Spheres from MDA-MB-231 Cells
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MDA-MB-231 cells were cultured in serum-free RPMI 1640, supplemented with B27 (Invitrogen) and 20 ng/ml bFGF (BD Biosciences, Franklin Lakes, NJ, USA), and 4 μg/ml heparin (Sigma Aldrich, St. Louis, MO, USA) and plated at 15,000 viable cells/well in ultralow attachment 6-well plates (Corning Incorporate, NY, USA). Complete serum-free medium was added to the cells every 3 days. Primary tumourspheres were enumerated at day 10. Primary tumourspheres were collected, and were enzymatically dissociated into single cells, re-plated in ultralow attachment 6-well plates (Corning Incorporate) at a density of 1,000 viable cells/well and enumerated at day 10.
3
Generating Anoikis-Resistant Cell Lines
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To establish the anoikis-resistant model, we cultured the MTH and U2OS cells in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA) for 3 days, transferred to normal plates with attachment culture until they formed monolayers, cultured again in ultra-low attachment 6-well plates for 14 days, and finally transferred to normal plates; the re-adherent cells were considered anoikis-resistant cells, and were designated MTHar and U2OSar, respectively (21 (link)). An inverted light microscope (Olympus Corp., Tokyo, Japan) was used to observe the cell morphology.
4
Mammosphere Formation and Cancer Stem Cell Enrichment
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Primary mammospheres were generated from MDA-MB-231 (Mesenchymal-like, MSL) or MDA-MB-468 (Basal-like, BL1) cells using MammoCult Human Medium Kit (STEMCELL Technologies). Briefly, after 7 days incubation on ultra-low attachment 6-well plate (Corning) in MammoCult growth medium, P1 mammospheres were collected by gentle centrifugation and then trypsinized for single cell suspension. Cells (10,000/well) were plated on ultra-low attachment 6-well plate (Corning) with complete MammoCult growth medium in the presence of GSI PF-03084014 (5 μM) or AKT inhibitor MK-2206 (5 μM) or IKK-targeting NF-κB inhibitor Bay11-7082 (1 μM) (Selleckchem, Texas) twice/week. Following 7 days of incubation, mammospheres (>100 μm) were counted manually using a Nikon Eclipse Microscope. For CSCs enrichment, CD44+CD24low and CD90+ cells were sorted from MDA-MB-231 cells using LSRII Flow cytometer (BD Biosciences). Sorted cells were plated for mammosphere formation, followed by treatments with GSI, AKT inhibitor or NF-κB inhibitor as described.
5
Embryoid Body Formation from iPSCs
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To induce embryoid body (EB) formation, iPS cells at day 6–7 after cell passage were treated with collagenase IV. Clumps were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Invitrogen) supplemented with 1% penicillin/streptomycin, 1 mM L-glutamine, 15% fetal calf serum (FCS, Invitrogen, Paris, France), 450 μM monothioglycerol, 50 μg/mL ascorbic acid (Sigma Aldrich, Paris, France) and 200 μg/L transferrin (Sigma Aldrich Paris, France), and supplemented with hematopoietic cytokines: 100 ng/mL stem cell factor (SCF), 100 ng/mL fms-like tyrosine kinase 3 ligand (Flt-3L) and 50 ng/mL thrombopoietin (TPO) (all from Peprotech, Thermofisher, Paris France). ESC and iPSC-derived EB were cultured in ultra-low attachment 6-well plates (Costar, Paris, France) for 16 days. Media was changed two or three times depending on EB proliferation. All cultures were incubated at 37 °C in 5% CO2.
6
Spheroid Formation Assay in 3D
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Cells were seeded into ultralow attachment 6-well plates (Corning Costar) and cultured in DMEM/F12 medium (Gibco) supplemented with 2% B27 (Life Technologies), 20 ng/ml FGF (R&D Systems, MN, USA), 20 ng/ml EGF (R&D Systems) and 5 μg/ml insulin (R&D Systems). Two weeks later, sphere pictures were obtained, and the sphere formation ratios were calculated.
7
Generation of Human Midbrain Organoids
8
Establishing Patient-Derived Organoids from Tumor Tissue
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PDOs were prepared according to Nickl et al.’s description [33 (link)]. For this purpose, fresh intraoperatively gained tumor tissue was temporarily stored on ice in Hibernate A medium. Necrotic areas and blood vessels were cleared and the tissue was minced carefully under the microscope into approximately 500 µm pieces with a scalpel. The tissue was then treated with RBC Lysis Buffer for 10 min and washed twice with Hibernate A medium. For incubation, tumor sections were transferred in PDO Medium consisting of 47.24% DMEM/F12, 47.25% Neurobasal, 0.02% B27 without Vitamin A (50×), 0.01% Glutamax, 0.01% N2, 0.01% NEAA, 0.004% penicillin/streptomycin, 0.001% β-Mercaptoethanol (all from Gibco, Carlsbad, CA, USA) and 0.00023% human insulin (Sigma Aldrich, St. Louis, MO, USA) to ultra-low attachment 6-well plates (Corning Costar, New York, NY, USA) and incubated at 37 °C, 5% CO2 and 95% humidity on an orbital shaker at 120 rpm. After 2 weeks of cultivation, PDOs formed successfully and could be used for further experiments [35 (link)]. At the end of the experiments, PDOs were fixed in 4% formalin (Carl Roth, Karlsruhe, Germany) for 24 h at 4 °C and then transferred to phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) for immunohistochemical staining.
9
Organoid Culture from Tumor Tissue
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Organoids were prepared according to the method described by Jacob et al. [24 (link)]. Fresh intraoperatively obtained tumor tissue was temporarily stored on ice in Hibernate A medium (Gibco, Carlsbad, CA, USA) (Figure 1 A). Next, the tissue was cleared of necrosis and blood vessels and carefully minced with a scalpel into approximately 0.5 mm pieces under the microscope (Figure 1 B–D). These pieces were then treated with RBC Lysis Buffer (Invitrogen, Carlsbad, CA, USA) for 10 min and washed two times with Hibernate A medium containing 1% Glutamax, 0.4% penicillin/streptomycin and 0.1% Amphotericin (HGPSA) (all from Gibco, Carlsbad, CA, USA). Sections were transferred into GBO medium consisting of 47.24% DMEM/F12, 47.25% Neurobasal, 0.02% B27 without Vitamin A (50×), 0.01% Glutamax, 0.01% N2, 0.01% NEAA, 0.004% penicillin/streptomycin, 0.001% β-Mercaptoethanol (all from Gibco, Carlsbad, CA, USA) and 0.00023% human insulin (Sigma-Aldrich, St. Louis, MO, USA) to ultra-low attachment 6-well plates (Corning Costar, New York, NY, USA) and incubated at 37 °C, 5% CO2 and 95% humidity on an orbital shaker at 120 rpm. After 2 weeks of culture, organoids formed successfully and could be used for further experiments (Figure 1 E–G).
10
Mammosphere Formation Assay with Digoxin
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After siRNA transfection for 24 h, cells were re-seeded at approximately 1 × 104 cells/mL in ultra-low-attachment 6-well plates (Corning Costar, Corning, NY, USA), and grown in suspension with serum-free DMEM/F12 medium (WelGENE) containing B27 (Gibco), 20 ng/mL epidermal growth factor (ProSpec, Rehovot, Israel), 20 ng/mL basic fibroblast growth factor (Gibco), 5 mg/mL insulin, 0.4% bovine serum albumin, and heparin (Sigma-Aldrich). Where indicated, digoxin was added to the medium after 9 days. After 10 days, mammospheres were visualized and counted using a microscope (Olympus IX71). 5-aza-2′-deoxycytidine (Sigma-Aldrich) was used at a concentration of 5 uM in a mammosphere formation assay.
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