I have TTX citrate ordered from Hello Bio. On using it at 1uM as my working concentration, it didn't show any effect.
How do I test whether the toxin is actually working or not?
What tissues or cells are you using it on? How are you assessing its effect? I've used it on isolated mouse skeletal muscle (i.e., soleus). It worked incredibly fast (i.e., in less than 30 seconds) and completely (i.e., contractile force reduced to zero). I used a bath concentration of 1 µM. However, it has been over 20 years since I did those experiments so I don't remember all of the details.
Dear Yojet,
I suppose you have patch clamp rig to measure ionic currents, if yes, then you can simply patch NSC derived neuron and record inward INa and apply TTX. You shall see rapid and almost complete block of sodium currents (see attached pic) which will confirm the efficacy and potency of TTX stocks you have. You can refer to the following paper for internal external combination and protocol to be used for recording.
Article Endothelin-1 Decreases Excitability of the Dorsal Root Gangl...
measuringEnsure that you aliquote your TTX and store it at -20/80 to preserves its efficacy. I hope it helps!
Best,
Nand
A straightforward alternative to patching would be by calcium imaging (e.g. with Fluo-4).
Hi Yojet,
Did you resolve your question? If it helps, I can provide you with some biological testing data for TTX citrate from Hello Bio, which was carried out in mouse cortical neurons (see protocol and data below).
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#Protocol 1: Effect of TTX citrate on action potentials in mouse cortical neurons
Whole cell voltage clamp recordings were obtained from layer V pyramidal neurons of the mouse prelimbic cortex brain slice.
Neurons were held at the resting membrane potential (~ -70 mV) and injected with a 500 pA 300 ms current step to induce action potential firing.
TTX was bath applied for 10 min first at 100 nM then 300 nM, 1 μM and 2 μM. After each drug application a current step was recorded to assess action potential blockade.
#Protocol 2: Effect of TTX citrate on EPSPs and action potential firing in mouse cortical neurons
Whole cell voltage clamp recordings were obtained from layer V pyramidal neurons of the mouse prelimbic cortex brain slice.
Neurons were held at the resting membrane potential (~ -70 mV) and EPSP were evoked by placing a stimulating electrode close to the recorded the neuron in layer II/III.
EPSPs and action potentials were evoked by single square (150 μs) pulse every 10 sec with an intensity that produced both an EPSP and action potential.
TTX was bath applied for 10 min first at 100 nM then 300 nM, 1 μM and 2 μM whist continually evoking and recording EPSP/A.Ps.
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If you are still having issues, please do contact the technical team at Hello Bio, who can talk to you in detail and will be more than happy to help!
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