Tang Proliferation/Mitosis - Dataset (ID:20004)

HMS Dataset ID: 20004
Dataset Title: Tang Proliferation/Mitosis
Project Summary Page(s): lincs.hms.harvard.edu/tang-jbiomolscreen-2013
Screening Lab Investigator: Yangzhong Tang
Screening Principal Investigator: Tim Mitchison
Assay Description: Tang Proliferation/Mitosis: Dose response of anti-mitotic compounds in human cancer cell lines at 24, 48 and 72 hours to determine effect on cell proliferation and mitosis.
Assay Protocol: 1. On Day 1, seed ~2000-3000 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 Day 2, 3, and 4 (0, 24, and 48 hrs after pin transfer), add 3 uL of 100 uM EdU (diluted from 10 mM stock to growth medium) to each well.

4. On Day 3, 4, and 5 (24, 48, and 72 hrs after pin transfer), perform the following to the plate to which EdU was added 24 hrs ago:
(A) Prepare 2 fixation/permeabilization solution in PBS and pre-warm it in 37C water bath.
(B) Add 30uL of the pre-warmed fixation/permeabilization solution to each well of cells using benchtop WellMate, so that the final concentration of formaldehyde is 3.7% and the final concentration of Triton-100 is 0.2%. Immediately centrifuge the plates at 1000rpm at room temperature for 20 minutes, in a plate centrifuge, while the cells are being fixed and permeabilized.
(C) Prepare an EdU reaction cocktail in H2O that has:
100mM TBS,
2mM CuSO4,
2uM Alexa488-azide, and
2mM ascorbic acid.
(Add individual components to the cocktail in the above order.)
(D) Add 50 uL of the above freshly prepared EdU reaction cocktail to each well.
(E) Incubate the plate at RT for 40-50 mins.
(F) Wash the plate three times with PBS. After washing, the residual volume in each well should be ~20uL.
(G) Add 20uL of anti-MPM2 primary antibody (Millipore #05-368, final dilution 1:500) in blocking buffer (4% BSA and 0.2% Triton-100 in PBS) to each well. Incubate for 1hr at RT.
(H) Wash the plate three times with PBS.
(I) Add 20uL of Goat-anti-mouse Alexa fluor 568 (Invitrogen A11004, final dilution 1:500) in blocking buffer to each well. Incubate for 1hr at RT.
(J) Wash the plate three times with PBS.
(K) Add 20uL of Hoechst (final concentration 1 ug/mL) in PBS to each well. Incubate for 30min at RT.
(L) Wash the plate. Seal with a plate seal.
(M) Image the plates 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, and then it identifies bright spots in the Texas Red (MPM2) channel to score for mitotic cells. In a separate step, the program measures the average FITC (EdU) intensity for all the nuclei, and scores for actively proliferating cells (cells that are incorporating EdU) by applying an EdU threshold.

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. EdU positive cells: The percentage of actively proliferating cells that have average FITC (EdU) intensity above the EdU threshold.
c. Mitotic cells: The percentage of cells that are MPM2-positive.
d. Non-mitotic cells that are EdU-positive: The percentage of EdU-positive cells within the non-mitotic population.
e. Mitotic cells that are EdU-positive: The percentage of EdU-positive cells within the mitotic population.
Assay Protocol Reference: Reference for EdU labeling:
Salic A, Mitchison TJ. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA. 2008 Feb 19;105(7):2415-20.
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2011-10-11
Most Recent Update: 2016-08-24