Individual MCF-7/GFP spheroids were formed in ultra-low adherent, Nunclon™ Sphera™ U-shaped-bottom, 96-well plate (Thermo Fisher Scientific). In brief, cells were detached from the cell culture flask using 1X TrypLE™ Express enzyme (Gibco) and re-suspended at a density of 104 cells/mL in pre-warmed complete minimum essential media (MEM) that contains 5 mg/mL human recombinant insulin (Gibco), 1X MEM non-essential amino acids (NEAA) (Gibco), 1 mM sodium pyruvate (Merck), and 50 mg/mL Kanamycin (BioConcept). 100 mL of cell suspension was loaded into each U-shaped-bottom well, resulting in an initial seeding of 1000 cells/spheroid. Cells were spun down at 250 g for 2 min and then kept in a humidified incubator at 37 °C and 5% CO2 (Binder GmbH) without medium exchange for 3 days. Before each experiment, MCF-7 spheroids were imaged with a Cell3iMager Neo plate scanning system (SCREEN Group) for quality check. Compact MCF-7/GFP spheroids with a diameter of approximately 350 mm were qualified for further experiment.
Kanamycin
Manufactured by BioConcept
Sourced in Switzerland
Kanamycin is an antibiotic used in laboratory settings. It functions as a selective agent, inhibiting the growth of bacteria that lack the necessary resistance mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Human recombinant insulin, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Calcium ionophore ionomycin, by Merck Group (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
2 protocols using kanamycin
1
MCF-7/GFP Spheroid Formation Protocol
2
Isolation and Cytokine Induction of Liver Lymphocytes
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Liver infiltrating lymphocytes and PBMCs were thawed in PBS with 10 μg/mL DNase I (Sigma-Aldrich, St. Louis, MO) to prevent cell clumping. The cells were resuspended in RPMI 1640 medium (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (Bioconcept, 4123 Allschwil, CH), 100 U/mL kanamycin, 2 mmol/L stable glutamine, 1 mmol/L sodium pyruvate, and 1% minimal essential medium (MEM) nonessential amino acids (all from Bioconcept), and 10 μg/mL DNase I. The cells were rested for 1 hour at 37°C before further processing. For intracellular cytokine induction, the cells were washed and resuspended in complete medium containing PMA (25 ng/mL), calcium ionophore ionomycin (1 μg/mL), and Brefeldin A (10 μg/mL; all from Sigma-Aldrich) for 4 hours. The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Norcross, GA) was used to identify viable cells before intracellular staining.
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