Jul 07, 2022
Deep Dye Drop Protocol
This protocol is a draft, published without a DOI.
- Chiara Victor1,
- Ben Gaudio1,
- Mirra Chung1,
- Mario Niepel1,
- Marc Hafner1,
- Luca Gerosa1,
- Clarence Yapp1,
- Kartik Subramanian1,
- Peter Sorger2,
- Caitlin Mills2,
- Ajit Johnson Nirmal2,
- Nicholas Clark2
- 1Harvard Medical School;
- 2Harvard University
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 Acknowledgement:
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
Optiprep ( Iodixanol)Sigma AldrichCatalog #D1556-250ML
Odyssey® Blocking Buffer (PBS) LI-CORCatalog #927-40000 927-40100
Microseal® ‘F’ Foil BioRad SciencesCatalog #MSF-1001
Hoechst 33342Catalog #H3570
Copper (II) sulfate pentahydrateSigma – AldrichCatalog #209198
Triton X-100 Sigma AldrichCatalog #X100
LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitationThermo FisherCatalog #L34974
1X PBSVWR ScientificCatalog #75800-986
Formaldehyde solution 37% Sigma AldrichCatalog #F1635-500ML
EdULumiprobeCatalog #10540
Sulfo-Cy3 azideLumiprobeCatalog #B1330
Phospho-Histone H3 Alexa 488 Cell Signaling TechnologyCatalog #3465S
Ascorbic acid SigmaCatalog #A4544
Stock solutions
- LIVE/DEAD Red- prepare according to directions (add 50 µL DMSO per tube), store at -20 °C , limit freeze/thaws
- EdU- prepare 10 millimolar (mM) working solution in DMSO, store at -20 °C 25 mg EdU 10 mL DMSO
- Sulfo-cy3 azide- prepare 4 Molarity (M) working solution in DMSO, store at -20 °C 3 mg sulfo-cy3-azide 1 mL DMSO
- Ascorbic acid- prepare 200 mg/ml working solution in water **fresh each time!**
- CuSO4.5H20- prepare 200 millimolar (mM) in water, store at room temperature, protected from light 250 mg CuSO4S 5 mL water
- Triton X-100- prepare 10% working solution in 1X PBS 10 mL Triton X-100 90 mL PBS
Pulse cells with EdU+ stain dead cells with LDR
For 10 ml:
1 mL optiprep (10% final)
9 mL PBS
5 µL LDR (1:2000 final)
10 µL EdU (10 µM final)
384-well plate: Add 15 µL per well along the edge of the wells using a multi-channel pipette and incubate for 01:00:00 (or desired pulse duration) @ 37 °C
96-well Plate: add 60 µL per well.
This file can be used for all solution calculations:
DDD.xlsx Fix cells
For 10 ml:
2 mL Optiprep (20% final)
6.9 mL PBS
1.1 mL formaldehyde (4% final)
384-well plate: Add 20 µL per well along the edge of the wells using a multi-channel pipette and incubate for 00:30:00 @ Room temperature in the dark (cover with foil)
96-well Plate: add 80 µL per well.
Run DeepDyeDrop1 protocol on the plate washer (aspirate all but 10 µL , dilute with 80 µL PBS, stop, store @ 4 °C . Aspirate all but 10 µL if continuing with permeabilization right away).
Permeabilize:
For 10 ml:
1 mL Optiprep (10% final)
8.5 mL PBS
500 µL 10% Triton X-100 (0.5 % final)
384-well plate: Add 15 µL per well along the edge of the wells using a multi-channel pipette and incubate for 00:20:00 @ Room temperature in the dark.
96-well plate: add 60 µL per well.
Click Reaction:
For 10 ml:
Combine *in order*
7 mL PBS
2 mL optiprep (20% final)
100 µL 200mM CuSO4 (2mMfinal)
10 µL 4mM sulfo-cy3-azide (4µM final)
1 mL 200 mg/ml ascorbic acid (20 mg/ml final)
384-well plate: Add 20 µL per well along the edge of the wells using a multi-channel pipette and incubate for 00:30:00 @ Room temperature in the dark.
96-well Plate: add 80 µL per well.
Run DeepDyeDrop1 protocol on the plate washer (aspirate all but 10 µL , dilute with 80 µL PBS).
Important note: Stopping point if not performing immunofluorescence staining. Include 2 µL Hoechst 33342 (1:5000 final) in the click reaction mix
Immunofluorescence:
384-well plate: Add 40 µL Odyssey blocking buffer per well and incubate @ Room temperature for 01:00:00 in the dark on a plate rocker at the slowest setting.
96-well plate: Add 160 µL per well.
Run DeepDyeDrop2 protocol on the plate washer (aspirate all but 10 µL )
Antibodies:
For 10 ml:
10 mL odyssey blocking buffer
2 µL Hoechst 33342 (1:5000 final)
5 µL pH3-A488 antibody (1:2000 final)
384-well plate: Add 15 µL antibody solution per well, incubate Overnight @ 4 °C in the dark, on a plate rocker at the slowest setting.
96-well plate: add 60 µL per well.
Run DeepDyeDrop3 protocol on the plate washer (wash once with PBST, twice with PBS, leave 80 µL PBS per well). Seal plates with Microseal 'F' foil seal and store @ 4 °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
- Apply flat field correction if available
- Segment nuclei based on Hoechst signal
- 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)
- calculate the nuclear area
- for each channel, calculate the average intensities within the nuclear mask, and within the surrounding ring
- for each channel, subtract the ring intensity from the nuclear intensity
- multiply the nuclear area by the Hoechst intensity for the DNA content
- calculate SER spot pixel size 8 texture feature for the LDR signal within the nuclear area
- output features (nuclear area,DNA content, Hoechst, EdU, pH3, LDR background corrected intensities, LDR texture feature)in a .tsv file
- identify cells in mitosis through pH3 intensity
- identify dead cells though LDR intensity and/or texture- threshold independent of cell line
- identify additional dead cells through size (small) and intensity (high)- optimize per cell line
- identify population of cells in each phase of the cell cycle based on DNA content and EdU intensity
- identify additional dead cells based on size (small) and intensity (high)
Data Analysis