Moerke 3 Color Apoptosis - Dataset (ID:20002)
|HMS Dataset ID:||20002|
|Dataset Title:||Moerke 3 Color Apoptosis|
|Project Summary Page(s):||lincs.hms.harvard.edu/tang-jbiomolscreen-2013|
|Screening Lab Investigator:||Nathan Moerke|
|Screening Principal Investigator:||Tim Mitchison|
|Assay Description:||Moerke 3 Color Apoptosis: Dose response of anti-mitotic compounds in human cancer cell lines at 24 and 48 hours to determine their effects on apoptosis and cell death. In this assay, the cell-permeable DNA dye Hoechst 33342 is used to stain the nuclei of all cells. The fluorescent caspase 3 reporter NucView488 stains the nuclei of cells undergoing apoptosis (in which caspase 3 is active), and the cell-impermeable DNA dye TO-PRO3 stains only the nuclei of dead or dying cells in which membrane integrity is compromised.|
Day 1: Seed cells in 384-well assay plates at approximately 2000 cells/well (the exact density varies by cell line), with 2 plates per cell line (one each for a 24 hr and 48 hr time point). Add 30 uL cell suspension per well.|
Day 2: Add compounds to plates by pin transfer.
Day 3: Process cells for 24 hr timepoint.
Prepare the 4X apoptosis reagent mixture fresh in PBS or media from stocks.
4X apoptosis reagent mixture composition:
(1) DEVD-NucView488 caspase substrate: 1 uM
(2) TO-PRO-3: 2 uM
(3) Hoechst 33342: 2 ug/mL
Add 10 uL mixture per well using WellMate plate filler or multichannel pipette, and leave in tissue culture incubator for 2 hrs.
Remove plate from incubator, seal, and image using IX Micro – should take 45-60 min to read an entire plate (assuming 10X magnification and 4 sites per well) depending on the exact settings.
If reading multiple plates, stagger reagent addition times by the time required for imaging so that the incubation times are equal.
Day 4: Repeat for 48 hr time point
Plates are imaged on the ImageXpress Micro screening microscope. 4 images are collected per well of the plate at 10X magnification, using the DAPI, FITC, and Cy5 filter sets of this instrument.
Images are analyzed using MetaXpress software. The multiwavelength cell scoring module of the software is used to detect all cells using the DAPI channel, score cells as apoptotic cells (NucView 488 positive) using the FITC channel, and score cells as dead or dying (TO-PRO-3 positive) using the Cy5 channel. The analysis produces for each well the total cell count, the % of cells in the well that are apoptotic, and the % of cells in the well that are dead or dying.
Hoechst 33342 (Invitrogen ) – 1 mg/mL stock in H2O, store at -20 degrees C
DEVD-NucView488 (Biotium) – 1 mM in DMSO, store at 4 degrees C and protect from light
TO-PRO-3 (Invitrogen) – 1 mM in DMSO, store at -20 degrees C
|Assay Protocol Reference:||
Cen H, Mao F, Aronchik I, Fuentes RJ, Firestone GL. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. FASEB J. 2008 Jul;22(7):2243-52.|
|HMS Dataset Type:||Microscopy/Imaging|
|Date Publicly Available:||2011-07-15|
|Most Recent Update:||2016-07-12|