Moerke 2 Color Apoptosis - Dataset (ID:20001)
|HMS Dataset ID:||20001|
|Dataset Title:||Moerke 2 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 2 Color Apoptosis: IA-LM, IST-MEL1, NCI-H1648, PC-9 and SK-LMS-1 cells. Dose response of anti-mitotic compounds in human cancer cell lines at 24, 48, and 72 hours to determine their effects on apoptosis. 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).|
Day 1: Seed cells in 384-well assay plates, at approximately 2000 cells/well (the exact density varies by cell line), with 3 plates per cell line (one each for a 24 hr, 48 hr and 72 hr time point). Add 30 uL cell suspension per well.|
Day 2: Add compounds to plates by pin transfer.
Day 3: Prepare cells for 24 hr timepoint.
Prepare the 4X apoptosis reagent mixture fresh in PBS
4X apoptosis reagent mixture composition:
(1) DEVD-NucView488 caspase substrate: 1 uM
(2) Hoechst 33342: 2 ug/mL
For 4 384-well plates, 25 mL of this mixture will be sufficient and allow plenty of dead volume.
For 25 mL of mixture add 25 uL of DEVD-NucView488 substrate (from refrigerator) and 50 uL of Hoechst 33342 (from freezer).
Use WellMate (hood or bench is fine) to add the mixture, using the designated manifold (labeled "NucView"). Add 10 uL per well, skipping the 1st 2 and last 2 columns. Spin plates briefly at 1000 rpm in plate centrifuge and put in incubator for 90 minutes.
Prepare 2X fixative solution: 2 % formaldehyde in PBS. Dilute a 36-36.5% formaldehyde stock bottle 1:20 in PBS. 100 mL fixative total is sufficient for 4 plates; add 5 mL formaldehyde stock to 95 mL PBS.
After 90 minutes, use benchtop (not hood) WellMate to add fixative to plates. Use the manifold labeled "Fixative". Add 40 uL per well (again skipping first 2 and last 2 columns). Spin plates briefly as before. Let fix 20-30 minutes at RT, then seal with metal foil, and image right away or store in the cold room until you are ready to image.
Day 4: Repeat for 48 hr time point
Day 5: Repeat for 72 hr time point
Plates are imaged on the ImageXpress Micro screening microscope (Molecular Devices). 4 images are collected per well of the plate at 10X magnification, using the DAPI and FITC 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 and score cells as apoptotic cells (NucView 488 positive) using the FITC channel. The analysis produces for each well the total cell count and the % of cells in the well that are apoptotic.
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 protected from light
|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|