Cell based assay nutlin 3

Example 4

SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range M will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 μM in 100 μL final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 l of 10× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Glo reagents according to manufacturer′ instructions.

Example 4

SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range μM will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 μM in 100 μL final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 μl of 10× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Glo reagents according to manufacturer' instructions.

Example 4

SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in 96-well plates a day prior to assay. On the day of study cells are washed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is added to cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks in sterile water, further dilutions made in 0.5% DMSO to keep the concentration of DMSO constant across the samples. The final concentration range μM will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0 μM in 100 μL final volume per well for peptides. Final highest DMSO concentration is 0.5% and will be used as the negative control. Cayman Chemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positive control. Nutlin was diluted using the same dilution scheme as peptides 10 μl of 10× desired concentrations is added to the appropriate well to achieve the final desired concentrations. Cells are then incubated with peptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere. Post-incubation period, cell viability is measured using Promega Cell Titer-Glo reagents according to manufacturer′ instructions.