Tang Mitosis/Apoptosis ver. II - Dataset (ID:20003)

HMS Dataset ID: 20003
Dataset Title: Tang Mitosis/Apoptosis ver. II
Publication(s) Using Dataset: PMID: 23788527
Project Summary Page(s): lincs.hms.harvard.edu/tang-jbiomolscreen-2013
Screening Lab Investigator: Yangzhong Tang
Screening Principal Investigator: Tim Mitchison
Assay Description: Tang Mitosis/Apoptosis ver.II: Dose response of anti-mitotic compounds in human cancer cell lines at 24, 48 and 72 hours to determine effect on apoptosis, mitosis and cell death.

In screening for small-molecule compounds that are effective at killing cancer cells, one-dimensional readout GI50, which is the EC50 value of growth inhibition, is usually used as the only criterion. A major problem with this one-readout approach is that other useful information is discarded, which could be critical for understanding the action of the compounds. In this screen, we use a single-cell-based imaging assay that can report multi-dimensional physiological responses in cells treated with small molecule kinase inhibitors. The data in this dataset are described in PMID: 23788527: Differential Determinants of Cancer Cell Insensitivity to Antimitotic Drugs Discriminated by a One-Step Cell Imaging Assay (J. Biomolecular Screening, 2013). The image analysis algorithm is available at https://github.com/xietiao/Tang_et_al_LINCS_cell_scoring.
Assay Protocol: 1. On Day 1, seed ~3000 cells in 30 uL of growth medium into each well of a 384-well clear-bottom black assay plate (Corning 3712), using a WellMate plate filler in a cell culture hood.

2. On Day 2, pin transfer performed by an ICCB-Longwood Screening Facility staff member using an Epson robot system. The pin transfer adds 100nL of each diluted compound from the 384-well compound library plate to each well of the assay plate.

3. On each day of Day 3, 4, and 5 (24, 48, and 72 hrs after pin transfer), perform the following:
(A) Prepare a cocktail of reagents in PBS that has 4ug/mL Hoechst33342 (Sigma B2261), 4uM LysoTracker-Red (Invitrogen L7528), and 2uM DEVD-NucView488 (Biotium 10403).
(B) Add 10uL of the reagent cocktail to each well of the assay plate using benchtop WellMate plate filler, so that the final concentration of Hoechst33342 is 1ug/mL, LysoTracker-Red is 1uM, and DEVD-NucView488 is 500nM.
(C) Incubate cells in a tissue culture incubator at 37C, 5% CO2 for 1.5 hrs.
(D) Prepare 2% formaldehyde in PBS and pre-warm it in 37C water bath.
(E) Add 40uL of the pre-warmed formaldehyde to each well of cells using benchtop WellMate, so that the final concentration of formaldehyde is 1%. Then immediately centrifuge the plates at 1000rpm at room temperature for 20 minutes, in a plate centrifuge, while the cells are being fixed.
(F) After 20 mins of fixation and centrifugation, seal the plates with adhesive plate seals.
(G) Image the plates, ideally within the same day, using the ImageXpress Micro screening microscope (Molecular Devices) and the 10x objective lens. Image 4 sites/per well.

Filter information:
DAPI [Excitation 377/50; Emission 447/60]
FITC [Excitation 482/35; Emission 536/40]
Texas Red [Excitation 562/40; Emission 624/40]

4. After image acquisition, image analysis is done using a customized Matlab program developed by Dr. Tiao Xie (Harvard Medical School). The program does segmentation on the DAPI channel to identify all nuclei, then it counts the bright, rounded cells in the Texas Red channel (LysoTracker-Red) to score mitotic cells. Finally it detects bright spots in the FITC channel (NucView) to score apoptotic cells. We also identify a population of late-stage dead cells with a "blurry" DAPI morphology, and no NucView Signal or LysoTracker Red signal. The MATLAB code for the image analysis algorithm is available at https://github.com/xietiao/Tang_et_al_LINCS_cell_scoring.

5. When reporting data, 5 parameters are reported for each replicate for each cell line and compound condition:
a. Cell Count: The total number of cells (nuclei) stained with Hoechst 33342 and detected in the DAPI channel.
b. Interphase cells: The total number of cells less the number of Apoptotic cells, Dead cells, and Mitotic cells.
c. Apoptotic cells: The cells stained with NucView.
d. Dead cells: The "late-stage" dead cells with blurry DAPI morphology that do not stain with either NucView or LysoTracker Red.
e. Mitotic cells: The cells that stain brightly with LysoTracker Red and that have a rounded morphology.
Assay Protocol Reference: Protocols and data are published in Tang Y, Xie T, Florian S, Moerke N, Shamu C, Benes C, Mitchison TJ. Differential Determinants of Cancer Cell Insensitivity to Antimitotic Drugs Discriminated by a One-Step Cell Imaging Assay. J Biomol Screen. 2013 Jun 20. PMID: 23788527.

Image analysis algorithm (MATLAB code) is available at https://github.com/xietiao/Tang_et_al_LINCS_cell_scoring

Reference for NucView:
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