Stock solutions of cocaine, isradipine, nomifensine, lidocaine, DHβE, CP8, L-741,626, and water-soluble cholesterol
Stephanie J Cragg, Katherine Brimblecombe
Abstract
This protocol details the making of the following stock solutions used in Brimblecombe, K.R. et al. (2023): cocaine (inhibits the activity of dopamine transporters (DATs)), isradipine (inhibits L-type voltage-gated Ca2+ channels (LTCC)), nomifensine (inhibits monoamine re-uptake), lidocaine (blocks voltage-gated Na+- channels), dihydro-β-erythroidine (DHβE) (nicotinic acetlycholine receptor (nAChR) antagonist), 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (CP8) (a potent and highly selective CaV1.3 L-type calcium channel antagonist; see Protocol: Synthesis of 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (CP8)), L-741,626 (inhibits D2 receptors), and water-soluble cholesterol (DAT function in Snca -null mice is augmented, see Threlfell et al., 2021).
Steps
Cocaine (5 µM)
Make 50 mL aliquots of 10 mM stock solution:
- add 3.4 mg per 1 mL dH2O
- store at -20°C
For each experiment, dilute 50 µL of 10 mM stock solution (one aliquot) in 100 mL of bicarbonate-buffered artificial cerebrospinal fluid (aCSF) solution for a final working concentration of 5 µM.
Isradipine (5 µM)
Make 50 µL aliquots of 10 mM stock solution:
- add 2.69 mL DMSO to 10 mg powder
- stored at -20°C
For each experiment, dilute 50 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 5 µM.
Nomifensine (10 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 3.54 mg per 1 mL 0.1 HCl
- stored at -20°C
For each experiment, dilute 100 µL of 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 10 µM.
Lidocaine (100 µM)
Make 100 µL aliquots of 100 mM stock solution:
- add 2.34 mg per 1 mL ethanol
- stored at -20°C
For each experiment, dilute 100 µL 100 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
Dihydro-β-erythroidine (DHβE)(1 µM)
Make 0.5 mL aliquots of 10 mM stock solution:
- add 2.8 mL dH2O to 10 mg powder
- store at -20°C
Make 100 µL aliquots of 1 mM working stock solution:
- add 4.5 mL dH2O to 0.5 mL 10 mM stock solution
- store at -20°C
For each experiment, dilute 100 µL of 1 mM working stock solution (one aliquot) in 100 mL of aCSF solution for a final working concentration of 1 µM.
CP8 (10 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 3.35 mg per 1 mL DMSO
- store at -20°C
For each experiment, dilute 100 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 100 µM.
L741-626 (1 µM)
Make 100 µL aliquots of 10 mM stock solution:
- add 2.93 mL DMSO to 10 mg powder
- store at -20°C
For each experiment, dilute 10 µL 10 mM stock solution (one aliquot) in 100 mL aCSF for final working concentration of 1 µM.
Water-soluble Cholesterol (50 µg/mL)
Make 45 mg of cholesterol per gram:
- add 11.1 mg water-soluble cholesterol to aCSF
Me-ß-cyclodextrin (1000 µM) - Control Solution
Make 20 µL aliquots of 0.5 M stock solution:
- add 655 mg per 1 mL dH2O [Yes it really is this soluble!]
- store at 4°C
For each experiment dilute 20 µL 0.5M stock solution (one aliquot) in 10 mL aCSF for final working concentration of 1 mM.
