Team:Evry/Experimentals Parameters


Protocols to determine the following experimental parameters

Auxin distribution in tadpole

To determine the site of auxin accumulation in tadpoles, intracardiac injection of auxin has to be done to ensure systemic distribution via the bloodstream. The next step is tadpole quenching followed by quantification and localization of auxin using commercially available fluorescent anti-auxin antibody.

Determination of auxin diffusing from tadpoles to the medium

To determine the coefficient of auxin diffusion from the skin to the medium (MMR), tadpole's skin is soaked in MMR+auxin medium for 20minutes. Next, the tissue is transferred to MMR auxin-free solution. Then , elevation of auxin level in MMR is measured. Finally, auxin level curve is plotted as auxin quantity as a function of time).

Determination of auxin diffusion from blood to muscle

To determine the coefficient of auxin diffusion from blood to muscle, intracardiac auxin injection is done. After 5min of tadpole recuperation in MMR, the tadpole is then euthanized, and muscles are extracted to identify auxin concentrations in different samples.

Determination of auxin half life in tissues (muscle and skin )

To determine the tissue half-life time of auxin, tissues were soaked in great MMR medium volume to recuperate auxin by diffusion.
Auxin concentration estimation in tissue was done by employing the massive volume difference between tissues and the bath.
Then place the tissues in a small volume of MMR with the same auxin concentration.
The volume of solution has to be equal to the volume of tissue.
Here we know how much auxin was present in the bath of a small volume.

After 5 minutes, determine the auxin presence in tissue and external environment.

Then the amount of auxin degraded by tissue volume in 5 minutes is calculated by deducting this value from the degradation of auxin in the MMR.

Repeat this experiment for at least 10 tadpoles, but with prolonged incubation time in the bath of small volume.
Finally just assign the value of auxin degradation over time to determine the parameters

Determination of auxin half-life in blood

Auxin half-life time in blood is determined by mixing a known amount of auxin with a defined volume of blood.Incubate for 5 minutes and determine auxin concentration.
Finally, deduct the initial quantity of auxin prior incubation from that measured after 5min incubation. Thus, auxin degraded in 5min is measured.
Repeat the experiment ten times, increasing the incubation time to trace the curve of auxin degraded in blood as a function of time.