Alamar Blue

Example 4

Cell-based using assays using human lens epithelial cell lines SRA 01/04 and B3 were also performed. The cells were exposed to UV or heat and cell viability assessed 24 hours post-exposure by Alamar blue staining. The results are shown in FIGS. 7A and 7B. In another experiment, SRA 01/04 cells were pre-incubated with varying concentrations of compounds two hours prior to UV exposure. Relative protection was measured as percent viability compared to vehicle control 24 hours following UV exposure. Effect of compounds from each of three distinct chemical series (macrocyclics, covalent, and catechol) are shown in FIG. 7B. Mean±SEM of the measurements are shown.

Ectopic expression of AAC-EGFP in B-3 cells shows that AAC forms inclusions that co-localize with p62 (arrows) (FIG. 8A). Automated image analysis of >2500 cells is shown in FIG. 8B. A statistically significant increase in GFP-positive inclusions due to AAC overexpression was observed. Mean±SEM of the measurements and p value (t test) are shown. The results show that high-content screening could be used to evaluate the cellular pharmacodynamic effects of SMDs for measuring cellular aggregates of AAC.

Example 57

Inhibitory activity on cell proliferation of representative compounds of the invention was determined using an Alamar Blue cell viability assay as described hereafter.

3000 cancer cells in 100 uL of cell culture media were plated in each well of a 96-well clear bottom, black wall cell culture-pretreated plate.

The next day compounds are serially diluted (3-fold in cell culture media) across a 96-well polypropylene mother plate from row A to row F, to yield 6 concentrations (10 uM, 3.3 uM, 1.1 uM, 370 nM, 124 nM and 41 nM) for each test compound. Rows G and H contain only DMSO.

Once titrations are made, the media in plates with cells were disposed and 100 μL of drug dilutions are transferred to plates with cells. After a ninety six-hour incubation at 37° C., 10 uL of resazurin solution from Alamar Blue Cell Viability kit (Invitrogen, Carlsbad, CA) was added to the media and cells were incubated at 37° C. for three more hours. At the end of this incubation the production of resofurin was measured using Spectrmax M2 microplate reader (Molecular Devices, Sunnyvale, CA)

Example 14

The docetaxel prodrug, synthesized as illustrated in FIG. 18A or B was administered to 2F2B cells in an alamar blue assay to measure metabolic activity. FIG. 22 shows a comparison between an αvβ3 targeted particle without docetaxel, an αvβ3 targeted particle with docetaxel, and an αvβ3 targeted particle with a docetaxel prodrug. As the assay progressed, the particle comprising the prodrug demonstrated a sustained suppression of cell metabolic activity, as compared to the targeted control without docetaxel.