Dimethyl Sulfoxide → Fluorescein 5-isothiocyanate

Beteiligte Stoffe

Reaktionsschritte

Jurkat cells were grown in RPMI 1640 media containing 25 mM Hepes and L-glutamine (Gibco) supplemented with 10% FCS and penicillin/streptomycin and harvested by centrifugation (200×g, 10 minutes) when they reached a concentration of 1×106 cells/mL. 1×109 Jurkat cells were resuspended in 100 mL RPMI 1640 media with 0.5% FCS. Cells were then treated with either 1% DMSO or 5 μM 5-[(5-gambogylaminopentyl)-thioureidyl]-fluorescein (Example 10) in DMSO for 30 minutes at 37° C. Cells were washed one time with 40 mL phosphate buffered saline (PBS) and lysed in 8 mL RIPA buffer (10×RIPA supplied by Upstate) and 0.1% Protease Inhibitor Cocktail (Sigma). The lysed cells were spun at 20,000×g for 10 minutes and the supernatant collected and referred to as “Jurkat lysate.”

Herkunft & Nachweise