5-[[2-(Diethylamino)ethyl]amino]-8-methoxy-6H-imidazo[4,5,1-de]acridin-6-one → HCID 131731139, compound

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Reaction Steps

A549 cells were grown on coverslips in 24-well plates until confluence was reached. Cells were then incubated with 10 μM C-1330 for 30 minutes and then exposed to light for 1 minute, in order to achieve lysosomal photodestruction. Control cells were loaded with C-1330 but were not illuminated. C1330-loaded cells were ether fixed and stained with Texas Red®-X Phalloidin (Life Technologies) with no illumination or stained with Texas Red®-X Phalloidin 2 hours after illumination. Cells were then washed twice with PBS and fixed by adding 4% formaldehyde for 10 minutes at room temperature. Following two additional washes with PBS, 0.1% Triton X-100 was added for 10 minutes, and then washed twice to remove the Triton X-100. Blocking was then achieved using a solution of 20% skim milk and 2.5% fetal calf serum for 20 minutes. At this stage, 5 μl of Texas Red®-X Phalloidin stock solution was added to 0.2 ml of PBS and the samples were incubated for 20 minutes while being protected from light. The samples were finally rinsed to remove excess Texas Red®-X Phalloidin. Coverslips were removed gently from wells and attached onto slides using Fluoromount-G. Fluorescent cells were analyzed by a fluorescence microscope, using an excitation wavelength of 545 nm and an emission wavelength of 605 nm.

Provenance & Evidence