MLPCN Ras selective lethality-BJeLR viability
Brent Stockwell, Columbia University, 614 Fairchild Center, MC 2406, 1212 Amesterdam Ave, New York, NY 10027, firstname.lastname@example.org, 212.854.2948 ..more
BioActive Compounds: 516
Depositor Specified Assays
Broad Institute MLPCN Ras Selective Lethality Project
Project ID: 2013
Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death
Brent Stockwell, Columbia University, 614 Fairchild Center, MC 2406, 1212 Amesterdam Ave, New York, NY 10027, email@example.com, 212.854.2948
Dan Zaharevitz, NCI Science Officer, firstname.lastname@example.org
The goal of the project is to identify small molecules in the MLSMR and related compound synthesized through follow-up chemistry that are selectively lethal to tumor cells expressing RAS oncogenes. These probes will not necessarily interact with the RAS pathway, but may induce cell death by affecting cellular processes that, in conjunction with the activation of the RAS pathway, selectively kill the tumor cells.
Cell viability of BJeLR (BJ fibroblasts transformed with hTERT, genomic SV40 LT and ST oncoproteins, and oncogenic HRASV12). Using CellTiterGlo, which measures the amound of intracellular ATP from living cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for 48 hours to allow detection of compounds that may be slower-acting.
Expected Outcome: Compounds that are toxic to these transformed fibroblasts, either selectively due to the HRAS oncogene or due to nonspecific toxicity, will cause a loss of luminescence signal due to fewer live cells. Specificity will be determined in future counterscreening.
Taken from 2013-01-A01-07:
Positive control: RSL3, from Stockwell lab, 32 mg, assigned BRD-K56411643-001-01-8
1. BJeLR cells (Stockwell lab) are normally maintained in 35mL of media in a T175 cell culture flask, which is kept in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
2. To passage, harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
3. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting. Cell density should be between 1.5 and 2 x 106/mL.
4. Transfer 4.5 x 10^6 (~2 mL) of the 13 mL cells in trypsin/media to 33 mL fresh media in a T175 for harvest at ~90% confluence two days later, or 1.5 x 10^6 (~1 mL)34 mL fresh media for harvest three days later.
5. Lines are stopped at P20 and restarted from an earlier frozen aliquot.
1. Harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
2. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting.
3. Add media to adjust the concentration of the cells to 40,000/mL and add a sterile stir bar to the flask.
4. Plate the cells to 384-well plates using the Combi (walkup room), transferring 25 uL to each well, for a total of 1,000 cells/well. Keep the cell flask on a stir plate, gently spinning, to maintain a uniform cell suspension during the plating operation.
5. Incubate the plates overnight in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
6. Transfer the plates to an online Liconic incubator at 95% humidity, 5% CO2, 37 degrees C.
7. Pin with 50 nL compound (BL2+ system, one dip) using 25nL head programmed to deliver 50 nL.
8. Return plates to the Liconic incubator at 95% humidity, 5% CO2, 37 degrees C for overnight incubation.
9. Transfer the plates to offline TC incubators, 95% humidity, 5% CO2, 37 degrees C. (This step is done to free up working space in the Liconic incubator.)
10. Move plates to racks in a Liconic incubator (on GS system), 95% humidity, 5% CO2, 37 degrees C. Load plate racks so that all plates will be pulled for the read run in the same order that they were pinned.
11. Initiate the automation protocol to start the read run. Plates are moved from the Liconic incubator to the GS carousel at normal room conditions (ambient humidity, 22 degrees C). Plates are allowed to cool on the carousel for 30 minutes, lids on.
12. Each plate is delided and 25 uL Cell Titer Glo (prepared as 1:3 dilution) is added to each well using the Combi.
13. Plates are incubated an additional 10 minutes in the carousel without lids.
14. Plates are read with the Envision, luminescence detection, read time = 0.1 second.
15. Plates are relided and discarded.
This assay belongs to specific project (2013, MLPCN Ras selective lethality) at the Broad Institute of MIT and Harvard
Activity_Score: plate effect corrected, background subtracted % activity average of two replicates
Activity_Outcome = 1
Activity_Score inhibition <50%.
Activity_Outcome = 2
Activity_Score inhibition >50%.
Activity_Outcome = 3
Wells where Activity_Score > 50% presumably due to uncorrected plate effects
Activity_Outcome = 4
Data Table (Concise)