A431-AD and CaSki-AD cells were treated for 24h and 48h with D609 (50 μg/ml) as described, collected and seeded in MEGM BulletKit serum free (Lonza) at 1 cell/well in 96 wells low-attachment plate (Corning). After one week, the number of spheres was counted and the sphere forming efficiency (SFE) evaluated as previously reported [5 (link)], using the formula: [number of spheres/number of seeded cells] x 100.
Low attachment plate
Manufactured by Corning
The Low Attachment Plate is a laboratory equipment designed to prevent cell adhesion. It features a surface treatment that minimizes cell attachment, allowing for the cultivation of suspension cultures and the study of anchorage-independent cell growth.
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Lab products found in correlation
Rock inhibitor y 27632, by Enzo Life Sciences (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Knockout serum replacement (ksr), by STEMCELL (1 mentions)
6 well plate, by Corning (1 mentions)
Sb431542, by STEMCELL (1 mentions)
Glutamax, by STEMCELL (1 mentions)
Non essential amino acids (neaa), by STEMCELL (1 mentions)
Aggrewell, by STEMCELL (1 mentions)
Ldn193189, by STEMCELL (1 mentions)
5 protocols using low attachment plate
1
Sphere Formation Assay for Cancer Stem Cells
2
EBV+ Nasopharyngeal Carcinoma Spheroids
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An EBV+ nasopharyngeal cell line (NPC43)20 (link) was used in this study. NPC43 cells, stably expressing Lifeact-mCherry,4 (link) were cultured in RPMI medium 1640 (1X, Gibco) supplemented with 10% fetal bovine serum, 1% antibiotic antimycotic (Gibco; 100 units per ml penicillin G sodium, 100 mg ml−1 of streptomycin, and 0.25 mg ml−1 of amphotericin B), and 0.2% 2 mM rock inhibitor Y-27,632 (ENZO) at 37 °C in a 5% CO2. NPC43 cells were maintained in 2D culture, passaged every 3 days in 1 : 3 ratio until 80% confluence. Cells cultured for more than 20 passages were discarded. To generate NPC tumour spheroids, NPC43 cells were harvested by trypsinization and resuspended in ice-cold tumour spheroid medium (cell culture medium containing 2% (v/v) Matrigel (CORNING, cat no. 354270)) at a concentration of 106 cells per ml. 100 μl of the cell suspension was mixed in 2.9 ml of cold tumour spheroid medium and seeded on low attachment plate (CORNING, cat no. 3471) at 37 °C in a 5% CO2. Spheroids were harvested after 4 days for dissemination assays but were observed for up to 7 days in some experiments.
3
Spheroid Formation and Modulation
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Cells were seeded in a low attachment plate (Corning), specific for spheroid formation; 2000 cells were plated, and growth was assessed after eight days by microscopy (Evos, inverted microscope). Cells were treated after 24 h of culture with ATP (250 µM) and adenosine (250 µM). Image analysis was performed using ImageJ software to determine the area growth of the spheroids.
4
Sphere Formation Assays for Cancer Stem Cells
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For sphere formation assays, 250 cells of A549, 500 of HCC827, and 1000 of H1975 were seeded in low‐attachment plate (Costar) with cancer stem cell (CSC) medium (RPMI1640 with 20 ng mL−1 EGF, 20 ng mL−1 FGF, 40 ng mL−1 IGF, and 1 × B27) for 10 days. Harvested spheres were dissociated by trypsin and re‐seeded using the same settings as the first generation.
5
Forebrain Organoid Generation from iPSCs
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Forebrain organoids were generated as previously described60 (link). On Day 0, iPSC colonies were detached by ReLeSR and aggregated to form EBs by Aggrewell (STEMCELL Technologies, #34811). The following day, EBs were resuspended and transferred to 6-well plate (Corning Costar) rotating at 110 rpm, containing DMEM/F12, 20% KnockOut Serum Replacement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM LDN193189, 5 μM SB-431542 and 2 μg/mL heparin (STEMCELL Technologies). On Day 6, half of the medium was replaced with induction medium consisting of DMEM/F12, 1X N2 Supplement, 1X Non-essential Amino Acids, 1X GlutaMax, 1 μM CHIR99021 and 1 μM SB-431542. On Day 7, organoids were embedded in Matrigel and cultured stationarily in Low-attachment plate (Corning Costar) in the induction medium. On Day 14, organoids were mechanically dissociated from Matrigel by manual pipetting in a 10 mL pipette tip. Organoids were transferred to 6-well plate rotating at 110 rpm, containing differentiation medium consisting of DMEM:F12/Neurobasal, 1X N2 and 1X B27 Supplements, 1 × 2-Mercaptoenthanol, 1X Non-essential Amino Acids, and 2.5 mg/ml human Insulin. From Day 35 to Day 60, 1% Matrigel was supplemented in differentiation medium.
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