Jan 18, 2024
Preparation of Holding ACSF (artificial cerebrospinal fluid) for slice electrophysiology
- 1University of California San Diego
Protocol Citation: Sarthak M. Singhal, Thomas Hnasko 2024. Preparation of Holding ACSF (artificial cerebrospinal fluid) for slice electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm6ool5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 26, 2023
Last Modified: January 18, 2024
Protocol Integer ID: 92735
Abstract
Hnasko lab- preparation of Holding ACSF
Holding ACSF is used for incubating brain slices prior to cell-attached or whole-cell electrophysiological recordings of neurons.
Materials
NaCl (Fisher Scientific, cat# BP358)
KCl (Fisher Scientific, cat# BP366)
NaH2PO4 (Fisher Scientific, cat# BP329)
D-glucose (Sigma Aldrich, cat# G8270)
CaCl2 (Fisher Scientific, cat# BP510)
MgSO4 (Fisher Scientific, cat# M80)
NaHCO3 (Fisher Scientific, cat# BP328)
Thiourea (Sigma Aldrich, cat# T8656)
Na-Ascorbate (Sigma Aldrich, cat# A7631)
Na-Pyruvate (Sigma Aldrich, cat# P5280)
Prepare 10X stock solution.
Composition of 1 L of 10X Holding ACSF (in mM): 1150 NaCl, 25 KCl, 12.3 NaH2PO4, 100 d-glucose, 20 CaCl2 and 20 MgSO4.
Composition of 1 L of 10X Holding ACSF (in g): 67.2 NaCl, 1.86 KCl, 1.47 NaH2PO4, 18.02 d-glucose, 2.94 CaCl2 and 4.93 MgSO4.
Dissolve all salts in 1 L of ddH2O to make 10X stock solution of Holding ACSF.
Dilute 10X solution to 1X for use in experiments.
Dilute 100 mL of 10X solution to ~ 800 mL with ddH2O and add (in mM): 26 NaHCO3, 2 thiourea, 5 Na-ascorbate and 3 Na-pyruvate; (in g): 2.18 NaHCO3, 0.15 thiourea, 0.98 Na-ascorbate and 0.33 Na-pyruvate.
Add ddH2O to final volume of 1 L .
Bubble the solution with carbogen (95% O2 5% CO2) for ~10 minutes.
After bubbling, measure and adjust pH of solution to ~ 7.3 with 12 N HCl (<1 ml).
Refrigerate until use.