Jul 07, 2022

Public workspaceDeep Dye Drop Protocol

This protocol is a draft, published without a DOI.
  • 1Harvard Medical School;
  • 2Harvard University
  • Laboratory of Systems Pharmacology
Caitlin Mills
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Chiara Victor, Ben Gaudio, Mirra Chung, Mario Niepel, Marc Hafner, Luca Gerosa, Clarence Yapp, Kartik Subramanian, Peter Sorger, Caitlin Mills, Ajit Johnson Nirmal, Nicholas Clark 2022. Deep Dye Drop Protocol. protocols.io https://protocols.io/view/deep-dye-drop-protocol-96zh9f6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: December 05, 2019
Last Modified: July 07, 2022
Protocol Integer ID: 30649
Keywords: High-throughput microscopy, live-cell assays, dose response, small molecule perturbation, viability, cell cycle, GR metrics
Funders Acknowledgements:
NIH
Grant ID: U54-CA225088
NIH
Grant ID: U54-HL127365
NIH
Grant ID: U24-DK116204
Abstract
High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo®). Here, a public domain “Dye Drop” method that uses sequential density displacement and microscopy to perform cell count and viability assays is described. Cell viability and DNA replication assays are followed by immunofluorescence imaging to collect single-cell dose-response data in the "Deep Dye Drop" version of the protocol. The resultant data can be used to calculate growth rate inhibition (GR) values and metrics. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.
Materials
MATERIALS
ReagentOptiprep ( Iodixanol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D1556-250ML
ReagentOdyssey® Blocking Buffer (PBS) LI-CORCatalog #927-40000 927-40100
ReagentMicroseal® ‘F’ Foil Bio-Rad LaboratoriesCatalog #MSF-1001
ReagentHoechst 33342Catalog #H3570
ReagentCopper (II) sulfate pentahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #209198
ReagentTriton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100
ReagentLIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitationThermo FisherCatalog #L34974
Reagent1X PBSVWR International (Avantor)Catalog #75800-986
ReagentFormaldehyde solution 37% Merck MilliporeSigma (Sigma-Aldrich)Catalog #F1635-500ML
ReagentEdULumiprobeCatalog #10540
ReagentSulfo-Cy3 azideLumiprobeCatalog #B1330
ReagentPhospho-Histone H3 Alexa 488 Cell Signaling TechnologyCatalog #3465S
Reagent Ascorbic acid Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4544

Stock solutions

  • LIVE/DEAD Red- prepare according to directions (add Amount50 µL DMSO per tube), store at Temperature-20 °C , limit freeze/thaws
  • EdU- prepare Concentration10 millimolar (mM) working solution in DMSO, store at Temperature-20 °C Amount25 mg EdU Amount10 mL DMSO
  • Sulfo-cy3 azide- prepare Concentration4 Molarity (M) working solution in DMSO, store at Temperature-20 °C Amount3 mg sulfo-cy3-azide Amount1 mL DMSO
  • Ascorbic acid- prepare Amount200 mg/ml working solution in water **fresh each time!**
  • CuSO4.5H20- prepare Concentration200 millimolar (mM) in water, store at room temperature, protected from light Amount250 mg CuSO4S Amount5 mL water
  • Triton X-100- prepare 10% working solution in 1X PBS Amount10 mL Triton X-100 Amount90 mL PBS
Pulse cells with EdU+ stain dead cells with LDR
For 10 ml: Amount1 mL optiprep (10% final) Amount9 mL PBS Amount5 µL LDR (1:2000 final) Amount10 µL EdU (10 µM final) 384-well plate: Add Amount15 µL per well along the edge of the wells using a multi-channel pipette and incubate for Duration01:00:00 (or desired pulse duration) @ Temperature37 °C
96-well Plate: add Amount60 µL per well.

This file can be used for all solution calculations:
Download DDD.xlsxDDD.xlsx


Fix cells

For 10 ml: Amount2 mL Optiprep (20% final) Amount6.9 mL PBS Amount1.1 mL formaldehyde (4% final) 384-well plate: Add Amount20 µL per well along the edge of the wells using a multi-channel pipette and incubate for Duration00:30:00 @ TemperatureRoom temperature in the dark (cover with foil)
96-well Plate: add Amount80 µL per well.
Run DeepDyeDrop1 protocol on the plate washer (aspirate all but Amount10 µL , dilute with Amount80 µL PBS, stop, store @ Temperature4 °C . Aspirate all but Amount10 µL if continuing with permeabilization right away).
Permeabilize:

For 10 ml:
Amount1 mL Optiprep (10% final)
Amount8.5 mL PBS
Amount500 µL 10% Triton X-100 (0.5 % final)

384-well plate: Add Amount15 µL per well along the edge of the wells using a multi-channel pipette and incubate for Duration00:20:00 @ TemperatureRoom temperature in the dark.
96-well plate: add Amount60 µL per well.
Click Reaction:

For 10 ml: Combine *in order* Amount7 mL PBS Amount2 mL optiprep (20% final) Amount100 µL 200mM CuSO4 (2mMfinal) Amount10 µL 4mM sulfo-cy3-azide (4µM final) Amount1 mL 200 mg/ml ascorbic acid (20 mg/ml final)
384-well plate: Add Amount20 µL per well along the edge of the wells using a multi-channel pipette and incubate for Duration00:30:00 @ TemperatureRoom temperature in the dark.
96-well Plate: add Amount80 µL per well.
Run DeepDyeDrop1 protocol on the plate washer (aspirate all but Amount10 µL , dilute with Amount80 µL PBS).

Important note: Stopping point if not performing immunofluorescence staining. Include Amount2 µL Hoechst 33342 (1:5000 final) in the click reaction mix

Immunofluorescence:

384-well plate: Add Amount40 µL Odyssey blocking buffer per well and incubate @ TemperatureRoom temperature for Duration01:00:00 in the dark on a plate rocker at the slowest setting.
96-well plate: Add Amount160 µL per well.
Run DeepDyeDrop2 protocol on the plate washer (aspirate all but Amount10 µL )
Antibodies:

For 10 ml: Amount10 mL odyssey blocking buffer Amount2 µL Hoechst 33342 (1:5000 final) Amount5 µL pH3-A488 antibody (1:2000 final)

384-well plate: Add Amount15 µL antibody solution per well, incubate DurationOvernight @ Temperature4 °C in the dark, on a plate rocker at the slowest setting.
96-well plate: add Amount60 µL per well.
Run DeepDyeDrop3 protocol on the plate washer (wash once with PBST, twice with PBS, leave Amount80 µL PBS per well). Seal plates with Microseal 'F' foil seal and store @ Temperature4 °C until imaging.
Image Acquisition:
On the Operetta:

Hoechst (ex 360-400 em 410-480) EdU-cy3 (ex 520-550 em 560-630) pH3-A488 (ex 460-490 em 500-550) LDR-A647 (ex 620-640 em 650-700)

On the IXM-C:

DAPI
FITC
TRITC
Cy5

Important note: Focus height will vary with plate type and cell line, but expect to image the pH3 higher than the others since cells ball up during mitosis
Image Analysis
  1. Apply flat field correction if available
  2. Segment nuclei based on Hoechst signal
  3. Define a ring around the nuclei, be sure this does not include any of the nuclear area (it will be used to subtract the local background from each channel)
  4. calculate the nuclear area
  5. for each channel, calculate the average intensities within the nuclear mask, and within the surrounding ring
  6. for each channel, subtract the ring intensity from the nuclear intensity
  7. multiply the nuclear area by the Hoechst intensity for the DNA content
  8. calculate SER spot pixel size 8 texture feature for the LDR signal within the nuclear area
  9. output features (nuclear area,DNA content, Hoechst, EdU, pH3, LDR background corrected intensities, LDR texture feature)in a .tsv file
  10. identify cells in mitosis through pH3 intensity
  11. identify dead cells though LDR intensity and/or texture- threshold independent of cell line
  12. identify additional dead cells through size (small) and intensity (high)- optimize per cell line
  13. identify population of cells in each phase of the cell cycle based on DNA content and EdU intensity
  14. identify additional dead cells based on size (small) and intensity (high)
Data Analysis