Reaction #57129

Reaction equation

ascorbic acid

pargyline

CO2

[3H]NE

HEPES

NaCl

KCl

CaCl2

MgSO4

glucose

→

desipramine

Solvents

DMSO

DMSO

Conditions

Workup

1. 1
   workup.WAITOn day 1, cells were plated at 3,000 cells
2. 2
   workup.ADDITIONPlates containing cells with 200 μl of assay
3. 3
   workup.ADDITIONto addition of compounds
4. 4
   workup.ADDITIONcontaining cells for a final tert concentration of 1 μM
5. 5
   workup.ADDITIONdiluted in assay
6. 6
   workup.ADDITIONTwenty-five microliters of assay buffer (maximum NE uptake) or tert compound were added directly
7. 7
   workup.ADDITIONcontaining cells in 200 μl of assay buffer
8. 8
   workup.WAITThe cells in assay buffer with tert compounds were incubated for 20 minutes at 37° C
9. 9
   workup.ADDITIONdiluted in assay buffer (120 nM final
10. 10
    Concentrationassay concentration)
11. 11
    workup.WAITthe plates were incubated for 5 minutes
12. 12
    Other(37° C.)
13. 13
    OtherThe reaction was terminated
14. 14
    Otherby decanting the supernatant from the plate
15. 15
    workup.ADDITIONThe plates containing cells
16. 16
    Washwere washed twice with 200 μl assay buffer (37° C.)
17. 17
    Otherto remove free radioligand
18. 18
    workup.WAITleft
19. 19
    Otherto dry for 2 minutes
20. 20
    Otherair-dried for an additional 10 minutes
21. 21
    Otherwere lysed in 25 μl of 0.25 N NaOH solution (4° C.)
22. 22
    workup.ADDITIONAfter cell lysis, 75 μl of scintillation cocktail was added to each well
23. 23
    Otherthe plates were sealed with film tape
24. 24
    workup.STIRRINGvigorously shaken for a minimum of 10 minutes
25. 25
    Otherto ensure adequate partitioning of organic and aqueous solutions
26. 26
    Otherto collect the raw cpm data

Procedure

On day 1, cells were plated at 3,000 cells/well in growth medium and maintained in a cell incubator (37° C., 5% CO2). On day 2, growth medium was replaced with 200 μl of assay buffer (25 mM HEPES; 120 mM NaCl; 5 mM KCl; 2.5 mM CaCl2; 1.2 mM MgSO4; 2 mg/ml glucose (pH 7.4, 37° C.)) containing 0.2 mg/ml ascorbic acid and 10 μM pargyline. Plates containing cells with 200 μl of assay buffer were equilibrated for 10 minutes at 37° C. prior to addition of compounds. A stock solution of desipramine was prepared in DMSO (10 mM) and delivered to triplicate wells containing cells for a final tert concentration of 1 μM. Data from these wells were used to define non-specific NE uptake (minimum NE uptake). Test compounds were prepared in DMSO (10 mM) and diluted in assay buffer according to tert range (1 to 10,000 nM). Twenty-five microliters of assay buffer (maximum NE uptake) or tert compound were added directly to triplicate wells containing cells in 200 μl of assay buffer. The cells in assay buffer with tert compounds were incubated for 20 minutes at 37° C. To initiate the NE uptake, [3H]NE diluted in assay buffer (120 nM final assay concentration) was delivered in 25 μl aliquots to each well and the plates were incubated for 5 minutes (37° C.). The reaction was terminated by decanting the supernatant from the plate. The plates containing cells were washed twice with 200 μl assay buffer (37° C.) to remove free radioligand. The plates were then inverted, left to dry for 2 minutes, then reinverted and air-dried for an additional 10 minutes. The cells were lysed in 25 μl of 0.25 N NaOH solution (4° C.), placed on a shake table and vigorously shaken for 5 minutes. After cell lysis, 75 μl of scintillation cocktail was added to each well and the plates were sealed with film tape. The plates were returned to the shake table and vigorously shaken for a minimum of 10 minutes to ensure adequate partitioning of organic and aqueous solutions. The plates were counted in a Wallac Microbeta counter (PerkinElmer) to collect the raw cpm data.

Source