Authors
Vibor Laketa
Image 1 Materials
stock(PIP3/AM)=50mM
Mr(PIP3/AM-DOG)=1471.16 g/mol
Mr(PIP3/AM-SAG)=1771.69 g/mol
example - 1mg of PIP3/AM-DOG is dissolved in 13.6µl of DMSO to obtain 50mM stock solution
use absolute, ultra pure DMSO (Sigma, Cat.#41647) (H2O presence is not good since it can induce PIP3/AM degradation during long-term storage)
20% pluronic F-127/DMSO (Invitrogen, Molecular Probes, Cat.# P-3000MP, )
Procedure
1. Before adding PIP3/AM to cells change the growth medium for medium with no serum (I normally use MEM with 30mM HEPES). The reson for this is that the esterases present in the serum may metabolise the AM ester groups and render PIP3/AM membrane impermeable
2. Mix PIP3/AM (stock) with 10% pluronic F-127/DMSO in 1:1 (v:v) ratio (for example 1µl of PIP3/AM (stock) with 1µl of 10% pluronic F-127/DMSO). Also 20% pluronic F-127/DMSO can be used (in which case 1:0.5 ratio should be used). Pluronic F-127 is commonly used with other reagents with AM esters, it should promote the solubilisation of PIP3/AM in physiological media. Next, take some medium from the cells (I typically take 50µl-100µl), resuspend the PIP3/AM/pluronic/DMSO mixture and immediatelly add everything to cells. Do this fast, leaving PIP3/AM in the cell medium for a longer time periods before adding it to cells may lead to its degradation. The concentration of PIP3/AM used on cells is between 10µM and 100µM.
Anticipated results
The PIP3/AM entry into cells is relativly fast (within minutes in HeLa and U 2-OS cells). PIP3/AM will distribute itself in most of the cellular membranes (ER, Golgi, PM)
The physiological effects are observed between 5min and 45min after PIP3/AM application (depending on the cell line, the kinetics of protective groups hydrolysis and the physiological process observed)
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