AID	Name	Type	Comment
1772	Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases	summary	Summary report for GTPase target project

University of New Mexico Assay Overview:
Assay Support: NIH 1R03 MH081231 01
Extended Characterization of Cdc42 Probe: Mechanism of Actions and Application to Cancer and Infectious Disease
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Implementation: Lin Hong, Ph.D.
Target Team Leader for the Center: Larry Sklar (lsklar@salud.unm.edu)
UNM Cheminformatics: Cristian Bologa, Ph.D., Oleg Ursu, Ph.D.

Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.

Assay Background and Significance:
Trypan blue exclusion from cells is a standard method of determining viability. This particular method determines the effect of 24 hour continuous exposure to a compound. Viability is determined using a Countess (Invitrogen) automatic cell counting device, which uses a slide of cells similar to a hemacytometer, and software that scans an image of the slide to determine live and dead cells.

This method will not detect early apoptosis, autophagy, or death after 3 days due to, for instance, a 1 hour exposure to 30 nM daunorubicin. Other methods to test cell viability include high throughput staining with propidium iodide alone (Edwards, BS, et al.; Current Protocols in Cytometry 9.24.1-9.24.9, 2007), staining with propidium iodide and fluorescein diacetate (Ross, DD, et al.; Cancer Research 49, 3776-3782, 1989), and measurement of ATP levels after cell lysis (Perez).

U937 cells are grown to a maximum of 0.5 million/mL in complete RPMI (including penicillin, streptomycin, glutamine and 10% by volume heat-inactivated fetal calf serum), and are resuspended in fresh medium at 1 million/mL. 1 microL of a compound in DMSO is added to 49 microL of medium, and 50 microL of cells is added to this and mixed. The suspension is transferred to a well of a sterile 96 well plate with a clear bottom (Costar 3596; Nunc 167008), and grown in a humidified CO2 incubator for 24 hours at 37 degrees C. The plate is mixed and checked under a microscope to ensure that the cells have not attached to the bottom, then the cell suspensions are transferred to tubes for ease of individual mixing. Each tube is then mixed just before removing 6 microL of suspension to a tube with 6 microL of trypan blue solution. This diluted suspension is mixed slowly by pipet around the tube in three places, and 10 microL of suspension is put into a slide to be read by the Countess. Viability is given in percent by the instrument: focus a control sample to obtain maximum viability, and fix the focus for the rest of the samples.

Calculations:

Viability is compared to control cells grown in 1 % DMSO vehicle, which averages 93 % viability. If the cells have a viability less than 85 % at 10 microM, the compound is considered active (cytotoxic). Or when looking at Ratio to DMSO control, value of < 0.85 is considered active (cytotoxic).
PUBCHEM_ACTIVITY_SCORE is based on cytotoxicity of the cell, hence it was calculated by the following equation:
SCORE = 100 - PercentViability
Active compounds have SCORE > 15

Viability_24_Hours_Continuous_Exposure_U937_Cells_Cdc42_ECP

Grant Number: NIH I RO3 MH081231-01