| Assay Method Information | |
| | Competition Binding Assay |
| Description: | For IC50 determination using Jurkat or Eμ-Myc1080 cells, samples containing 2×105 cells (or 4×105 cells; exact cell number used in the respective evaluations is given in the results section) in Hank's balanced salt solution (HBSS)/1% Bovine Serum Albumin (BSA) were incubated with 100.000 cpm of the respective radioligand (approx. 0.1 nM) in the presence of increasing concentrations (10−11 to 10−5 M) of the respective peptide of interest (n=3 per concentration). The total sample volume was 250 μL. After incubation at room temperature (RT) for 120 min with gentle agitation (200 mot/min), the tubes were centrifuged (5 min, 450g, Megafuge 1.0, Heraeus Thermo Scientific) and the supernatant was carefully removed. After washing twice with 400 μL of cold HBSS, the amount of cell-bound radioligand was quantified using a □-counter.[0403]In the case of the adherent C6 glioma cells, cells were harvested using Trypsin/EDTA (0.05 % and 0.02%) in PBS one day prior to the experiment, centrifuged and resuspended with culture medium to a concentration of app. 150.000 cells/mL. The suspension was transferred into PLL-coated twenty-four-well plates (1 mL/well, Greiner, Solingen. Germany) and placed in the incubator overnight. On the day of the experiment, the culture medium was removed and the cells were washed with 250 μL of HBSS before being left to equilibrate in 200 μL of HBSS (1% BSA) at 37° C. for a minimum of 15 min before the experiment. Then, cells were incubated with 100.000 cpm of the respective radioligand (approx. 0.1 nM) in the presence of increasing concentrations (10−11 to 10−5 M) of the respective compound of interest (n=3 per concentration). The total sample volume was 250 μL. After incubation at room temperature (RT) for 120 min, the supernatant was removed and cells were washed twice with 200 μL of cold HBSS. Then, cells were lysed using 250 μL of 1 N NaOH, the lysate was transferred to vials and combined with 250 μL of HBSS used for rinsing the wells. Quantification of the amount of free and bound activity was performed in a □-counter. |
| Affinity data for this assay | |
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