Reaktion #2306251

Reaktionsgleichung

[3H]-SCH 58261

[3H]-DPCPX

A1

NECA

NECA

→

Adenosine

Lösungsmittel

Reaktionsbedingungen

Aufarbeitung

1. 1
   workup.ADDITIONAdd 100 μl of diluted membranes
2. 2
   workup.ADDITIONcontaining the appropriate receptor
3. 3
   workup.ADDITIONMix
4. 4
   Filtrationfilter plates
5. 5
   workup.ADDITIONAdd 45 μl Microscint 20 (Packard), and count

Vorschrift

Perform assays in deep well 96 well plates. Total assay volume is 200 μl. Add 50 μl compound dilution buffer (total ligand binding) or 50 μl CGS 15923 working solution (A2a non-specific binding) or 50 μl NECA working solution (A1 non-specific binding) or 50 μl of drug working solution. Add 50 μl ligand stock ([3H]-SCH 58261 for A2a, [3H]-DPCPX for A1). Add 100 μl of diluted membranes containing the appropriate receptor. Mix. Incubate at room temperature for 90 minutes. Harvest using a Brandel cell harvester onto Packard GF/B filter plates. Add 45 μl Microscint 20 (Packard), and count using the Packard TopCount Microscintillation Counter. Determine IC50 values by fitting the displacement curves using an iterative curve fitting program (Excel). Determine Ki values using the Cheng-Prusoff equation.

Quelle