9/22/2004 Colin
TTX from
Alomone Labs, Israel. TTX w/ citrate.
Na channel toxin
1 mg per ampoule, MW =
319.28, soluble in dH2O
lyophilised = 1 year @ 4 deg
C / liquid = 6 months @ -20 deg C
desired final concentration
= 1 uM
1 uM in 100 ml external
solution = 0.031928 mg in 100 ml
1
mg TTX in 1566 ul of dH2O => 50 ul aliquots each of 0.031928 mg
aliquot
solution is concentration of 2 mM
Picrotoxin from
Tocris Cookson.
GABA A R channel toxin
1 g per ampoule, MW = 602.00,
soluble in DMSO
lyophilised = 1 year @
RT / liquid = 6 months @ -20 deg C
desired final concentration
= 100 uM
100 uM in 100 ml external
solution = 6.02 mg in 100 ml
500
mg PTX in 8306 ul (8.31 ml) of DMSO => 100 ul aliquots of 6.02 mg aliquot
solution is concentration of 100 mM
APV from
Tocris Cookson.
NMDA
R antagonist
10 mg per ampoule, MW =
197.13, soluble in dH2O
lyophilised = 6 months @
RT / liquid ~ 2
months @ -20 deg C
desired final concentration
= 100 uM
100 uM in 100 ml external solution
= 1.9713 mg in 100 ml
10
mg APV in 508 ul of dH2O => 100 ul aliquots each of 1.9713 mg
aliquot
solution is concentration of 100 mM
CNQX from
Tocris Cookson.
AMPA, Kainate antagonist
10 mg per ampoule, MW =
232.16, soluble in DMSO to 100 mM
lyophilised = 6 months @
RT / liquid ~ 2
months @ -20 deg C
desired final concentration
= 20 uM
20 uM in 100 ml external
solution = 0.46432 mg in 100 ml
10
mg CNQX in 1077 ul of DMSO => 50 ul aliquots each of 0.46432 mg
aliquot
solution is concentration of 40 mM
NBQX from
Tocris Cookson.
AMPA, Kainate antagonist
5 mg per ampoule, MW =
336.28
desired final concentration
= 20 uM
5
mg NBQX in 743 ul of DMSO => 100 ul aliquots each of 0.6726 mg
Kynurenic
from Sigma
Acid General
glutamate R antagonist (AMPA and NMDA Rs)
1 g per ampoule, MW = 189.2,
desired final concentration
= 2 mM
2 mM in 100 ml external
solution = 37.84 mg in 100 ml
Do
not make up stock and freeze. Instead, on day of experiment, dissolve
direct into external
solution and stir for 10-20 mins.
SR95531 from
Tocris (1262)
Hydrobromide
GABAA R Antagonist of choice (no effects on K+ channels)
(GABAzine) 10 mg ampoule, MW = 368.23, soluble
in dH2O
desired final concentration
= 3 uM
3 uM in 100 ml external
solution = 0.1105 mg in 100 ml
10
mg of SR95531 in 9ml dH2O => 90 aliquots of 100ul each 0.1105mg
1
mg of SR95531 in 900ul dH2O => 9 aliquots of 100ul each 0.1105mg
Each aliquot will give 3 uM
in 100 ml external
Bicuculline
from Sigma
Methiodide GABA A receptor
antagonist
100 mg pot, MW = 509.3,
soluble in dH2O to 1.8 mg/ml
[lyophilised > 6 months @
2-8 deg C, liquid ~ 2 months @ -20
deg C]
desired final concentration
= 100 uM
100 uM in 100 ml external
solution = 5.093 mg in 100 ml
100 mg Bicuculine Methiodide in 29452 ul (29.5 ml)
of dH2O => 1500 ul aliquots each of 5.093 mg, aliquot solution is
concentration of 6.666 mM
QX-314 from
Alomone Labs, Israel.
Blocks
Na channels from inside cell
500 mg ampoule, MW = 298.85,
soluble in dH2O or internal solution
store @ RT
desired final concentration
in patch pipette = 2-5 mM
dissolve ~1.5 mg in 100 ul internal solution, take
volume equivalent to 0.598 mg and add this to 400 ul of internal solution to
give 5 mM,
or take volume equivalent to 0.3 mg and add this to
200 ul of internal solution to give 5 mM
Bungarotoxin from Calbiochem.
nACHR antagonist
1mg ampoule, soluble in dH2O, store stocks @ -20 deg
C
desired final concentration = 1-2 ug per ml (Zhang
et al. 1998)
25 ml at this concentration = 50 ug in 100ml
Make 50ul aliquots of 1mg/ml in dH2O and add one 50
ul aliquot to 25 ml external to achieve 2 ug per ml
Tubocurarine from Sigma.
nACHR antagonist
desired final concentration = 50 uM (Zhang et al.
1998)
50 uM in 50 ml external solution = 3.409 mg in 100
ml
100
mg Tubocurarine in 5867ul dH2O => 100ul aliquots of 1.705 mg
each is enough to make 50 uM
of 50 ml external
Glutamic from
Tocris.
Acid Major
agonist for EAA receptors in CNS
1 g ampoule, MW = 147.13,
soluble to 1 eq NaOH
store @ RT, stock solutions
at –20 deg C
desired final concentration
= 200 uM
200 uM in 100 ml external
solution = 2.9426 mg in 100 ml
58.852 mg Glutamic Acid in 2000 ul (2 ml) of 200mM
NaOH => 100ul aliquots of 2.9426 mg, aliquot solution is concentration of
200 mM
Muscimol from
Sigma
Selective GABAA agonist
10 mg pot, MW = 114.1, soluble to 100mM in 1 eq NaOH
desired final concentration = 30 uM
30 uM in 100 ml external solution = 0.3423 mg in 100
ml
30 uM in 1 ml external solution = 0.003423 mg in 100
ml
10 mg Muscimol in 1460ul
NaOH => 50ul aliquots for 30 uM in 100ml
or
1ul in 2ml external to achieve 30uM
NMDA from
Tocris (0114).
NMDA R Agonist
50 mg ampoule, MW = 165.15,
soluble in dH2O
desired final concentration
for puffer application = 200 uM – 1 mM
desired final concentration
for bath application = 20 uM (Ben-Ari)
200 uM in 1 ml external
solution = 0.033 mg in 1 ml external
20 uM in 100 ml external
solution = 0.33 mg in 100 ml dH2O
5 mg NMDA in 1500 ul dH2O
=> 100ul aliquots of 0.2943 mg
50 ul of aliquot in 50 ml =
20 uM
10 ul of aliquot in 1 ml =
200 uM
aliquot solution is
concentration of 2 mM
GABA R agonist
1 g per ampoule, MW =
103.12, soluble in dH2O to 100 mM
lyophilised = 6 months @
RT / liquid ~ 2
months @ -20 deg C
desired final concentration
= 50 uM (see Kriegstein ref)
50 uM in 100 ml external
solution = 0.516 mg in 100 ml
10
mg GABA in 1938 ul of dH2O => 100 ul aliquots of 0.516 mg aliquot
solution is concentration of 50 mM