The sorted cells (SP or Aldhhigh) were plated in ultra-low attachment 96-well plate (Corning Inc.) at a density of 10,000 cells/ml (1000 cells/100 μl/well) in stem cell selective medium [Dulbecco’s modified Eagle’s medium:F12K (1:1) supplemented with N2 supplement (1×) (Invitrogen), 10 ng/ml EGF, and 10 ng/ml bFGF (Sigma Aldrich)] at 37°C for 10 days [25,26] . The spheres were observed using an automated Zeiss Observer Z.1 inverted microscope, and images were acquired using the AxioCam MRm3 CCD camera and Axiovision version 4.7 (Carl Zeiss Inc., Germany). The numbers of spheres greater than or equal to 50 μm were counted. To study the effect of the drugs on the self-renewal ability of SP cells, the appropriate concentrations were added to the respective wells on Day 0 and Day 5, and the size and number of the spheres were analyzed on Day 10. The sphere formation assays were performed twice with triplicates of each treatment in every assay.
N2 supplement 1
Manufactured by Thermo Fisher Scientific
The N2 supplement (1×) is a laboratory product designed to provide a source of nitrogen. It serves as a nutrient supplement for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Merck Group (2 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions)
Axiovision version 4, by Zeiss (1 mentions)
Observer z1 inverted microscope, by Zeiss (1 mentions)
Axiocam mrm3 ccd camera, by Zeiss (1 mentions)
Ultra low attachment 96 well plate, by Corning (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Dmem f12k medium, by Thermo Fisher Scientific (1 mentions)
Accutase reagent, by Innovative Cell Technologies (1 mentions)
Fumitremorgin c, by Merck Group (1 mentions)
Facsvantage, by BD (1 mentions)
2 protocols using n2 supplement 1
1
Sphere Formation Assay for Cancer Stem Cells
2
Isolation and Culture of Side Population Cells
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To isolate side population cells, asynchronously growing cells were harvested using Accutase reagent (Innovative cell technologies, Inc.), washed once with DPBS and re-suspended in DMEM:F12K medium (Gibco, Life Technologies) with 2% FBS at 1 ×106 cells/ml density. Cells were then incubated with 4 μg/ml of Hoechst 33342 dye (Life Technologies) for 90 min at 37 °C in the presence or absence of 1 μM Fumitremorgin C (Sigma Aldrich). The side population (SP) cells were sorted using FACS Vantage (BD FACSDiVa) cell sorter as described in previous publications17 (link),18 (link). Data analyses were done using the FlowJo software (Tree Star). The isolated cells were further used for various experiments.
To generate the SP Adherent (SPAdh) cells, sorted SP cells were grown on Poly-D-Lysine and Laminin coated plates (Sigma Aldrich) in stem cell selective media [DMEM:F12K (1:1) supplemented with N2 supplement (1×) (Invitrogen), 10 ng/ml EGF and 10 ng/ml bFGF (Sigma Aldrich)]17 (link). The HDAC11 inhibitor treatments on SPAdh cells were carried out at 2.5 μM concentrations for 96 hours and were further analyzed by RT-PCRs.
To generate the SP Adherent (SPAdh) cells, sorted SP cells were grown on Poly-D-Lysine and Laminin coated plates (Sigma Aldrich) in stem cell selective media [DMEM:F12K (1:1) supplemented with N2 supplement (1×) (Invitrogen), 10 ng/ml EGF and 10 ng/ml bFGF (Sigma Aldrich)]17 (link). The HDAC11 inhibitor treatments on SPAdh cells were carried out at 2.5 μM concentrations for 96 hours and were further analyzed by RT-PCRs.
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