Protocol to Prepare AP 20187 for In Vitro and In Vivo Experiments

This is intended as a guide only; for full experimental details please read the references provided below.

Resuspension protocol for in vitro and in vivo experiments

1. Prepare a stock solution of AP 20187 (Cat. No. 6297) in 100% ethanol. We recommend preparing a 42 mM stock solution for use with the protocol for in vivo working solution preparation (below).

2. Tightly seal the vial containing the solution. If necessary, vortex until completely dissolved.

This stock solution can be stored at –20°C.

NOTE: Keep the vial of AP 20187 stock solution tightly sealed. Leaving the vial open can result in evaporation of ethanol from the stock solution, which will alter the concentration and may cause precipitation issues when preparing the dosing solution.

Preparation of working solutions for dosing AP 20187 in vivo

Prepare the AP 20187 dosing solution within 30 min of administration. To conserve your stock solution, we recommend preparing only the volume required for each experiment. To administer 10 mg/kg in a dose volume of 4 mL/kg, a 1.7 mM (2.5 mg/mL) dosing solution of AP 20187 should be used.

To prepare 1 mL of 1.7 mM dosing solution:

1. Pipette 40 μL of 42 mM AP 20187 stock solution into a vial.

2. Add 100 μL PEG-400 (100%) and vortex. 

3. Add 860 μL Tween®-80 (2%) in water. Upon addition of Tween® 80, a cloudy solution may result, this is transient and clears after gentle agitation or vortex.

4. Administer within 30 min by IP or IV injection.

NOTE: The 1.7 mM dosing solution is near saturation concentration, slight alterations in preparation may cause precipitation. To prepare a dosing solution at a lower concentration, dilute the stock solution to the appropriate concentration using 100% ethanol and follow steps 1–4. This will maintain the final ethanol concentration of 4%.

NOTE: Substitution of Tween®-80 with Tween®-20 can cause solubility issues and is not recommended.

TWEEN is a registered trademark of Croda International PLC 

References

Jacobs et al (2018) StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins. Nat.Methods 15 523 PMID: 29967496.

Ono (2017) Exposure to sequestered self-antigens in vivo is not sufficient for the induction of autoimmune diabetes. PLoS One 12 e0173176 PMID: 28257518.

Baker et al (2016) Naturally occurring p16^Ink4a-positive cells shorten healthy lifespan. Nature 530 184 PMID: 26840489.

Shariat et al (2001) Adenovirus-mediated transfer of inducible caspases: a novel "death switch" gene therapeutic approach to prostate cancer. Cancer Res. 61 2562 PMID: 11289132.