TITLE: Deep Dye Drop Protocol
AUTHORS: 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

[DESCRIPTION]
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.

[STEPS]
1. 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 60 min (or desired pulse duration) @ 37 °C 
96-well Plate: add 60 µL  per well.
2. 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 30 min  @ Room temperature in the dark (cover with foil) 
96-well Plate: add 80 µL  per well.
2.1. 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).
3. 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 20 min  @ Room temperature in the dark.
96-well plate: add 60 µL  per well.
4. 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 30 min  @ Room temperature  in the dark. 
96-well Plate: add 80 µL  per well.
4.1. 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

5. Immunofluorescence: 

384-well plate: Add 40 µL  Odyssey blocking buffer per well and incubate @ Room temperature  for 60 min  in the dark on a plate rocker at the slowest setting. 
96-well plate: Add 160 µL per well. 
5.1. Run DeepDyeDrop2 protocol on the plate washer (aspirate all but 10 µL )
5.2. 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    @ 4 °C  in the dark, on a plate rocker at the slowest setting.
96-well plate: add 60 µL per well. 
5.3. 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. 
6. 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
7. 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)
1.1. 
This file can be used for all solution calculations: 
  


8. Data Analysis