K+ aspartate internal solution (w/ biocytin)

Based on the standard recipe for internal from the Wilson lab, updated by Yvette 10/2020

Note: to make Cs aspartate internal substitute KOH for CsOH

• ATP, GTP and Neurobiotin should be stored in the freezer (-20C) in a container w/ drierite.

** alternatively you can use neurobiotin instead of biocytin.  Do not use both!  It seems that neurobiotin can provide better fills in certain cell types, however there are also anecdotal observations of it making certain cell types unhealthy. 

Important notes before starting:

·  Perform a standard 3-point calibration on the pH meter

·  Verify the reverse osmosis machine resistance is 18.2 MΩ

·  Make 5-10 milliliters of KOH 1M stock solution fresh in a scintillation vial each time:

Note: KOH in contact with the moisture from your skin can cause corrosive burns, and can cause acute oral toxicity and serious eye damage. Thus, handle it in the fume hood, and wear PPE (nitrile gloves, long pants, and closed-toed shoes) and lab coat. Dispose of gloves in biohazard waste bins, and vials into a glass disposal container, and collect any excess KOH solution in a container at the satellite accumulation area in the fume hood.

1. Place 2-4 pellets directly to a tared scintillation on the balance, note the mass

2. Add Milipore water to the scintillation vial to yield a 1M stock

3. Shake or vortex to dissolve KOH pellets

·  If paranoid, make KCl 50mM stock solution fresh as well.

For making 5mL total volume refer to the middle columns (yellow):

1. Add 4.2 mL Milipore water in a clean scintillation vial.
2. Add KOH (from 1M stock solution) and aspartic acid (powdered) to scintillation vial.
3. Vortex and sonicate to put in solution. This may take several minutes.
4. If KOH and aspartic acid do NOT go into solution:

                                           i. add a few drops of KOH (1M stock),

                                          ii. pH to check that it’s still acidic,

                                         iii. vortex for several minutes,

                                         iv. repeat i-iii until all aspartic acid dissolves

1. Add HEPES, EGTA, and KCl (50mM stock)
2. Adjust pH to 6-8 by adding KOH or aspartic acid.
3. Add MgATP, Na3GTP, and NeuroBiotin or Biocytin Hydrazide
4. Once in solution, adjust the pH to ~7.2 (7.1-7.3 OK)
5. Add Milipore water until the osmolarity reaches ~265 mOsm (262-268 OK).
6. Aliquot using a syringe with a tip filter (Millipore Millex-GV, pore size 0.22 microns).
7. Switch to a fresh as the filters become clogged
8. Store aliquots at -20 °C

For making 20mL total volume refer to the rightmost (green) columns:

1. Add 16.8 mL Milipore water in a flat-bottomed container (e.g. a 150 mL disposable flask).

2. Follow instructions 2+ as above only using the rightmost (green) columns.

Important

This saline should be disposed of as hazardous waste. At the lab SAA (in the fume hood), collect left over saline in a 1 gallon white container. Attach a hazardous waste label to the container listing “trace ATP adenosine 5’-triphosphate magnesium, plastic tubes” and checking “Toxic.”