TITLE: 96-well plate OT-2 liquid handler integrated live-cell and endpoint viability drug activity screen
AUTHORS: Rolando DZ Lyles, M Julia Martinez, Benjamin Sherman, Kerry Burnstein

[DESCRIPTION]
This protocol details OT-2 liquid handler integrated live-cell and endpoint viability drug activity screening on a 96-well plate.

[BEFORE_START]
Before starting:

Select the drug to be tested and determine the max concentration to be tested.
Starting with the defined max concentration, this protocol tests a single drug at 6 concentrations (including a vehicle control) at 1:3 serial dilutions.

Determine the cell line models which will be tested. Expand cell lines to ~50-60% confluence in any standard tissue t75-Flask or 15cm tissue culture dish.

Determine cell-specific seeding density and media condition to be tested.

[GUIDELINES]
To overcome the individual shortcomings of live-cell imaging and endpoint viability, it is recommended to integrate both cell viability assessment methods in a single drug screen. This protocol is designed to evaluate drug efficacy simultaneously robustly and reproducibly across multiple cell lines through the integration of automation, live-cell imaging, and an endpoint metabolic ATP viability assay.

*It is recommended that this protocol be used after defining optimized assay-specific growth conditions through the application of the protocol “96-well plate cell growth optimization for integrated live-cell and endpoint viability drug screening assay” for each tested cell line.
 

This protocol is designed to simultaneously screen a single compound across a panel of cell lines in a semi-automated procedure integrating sequential live-cell imaging and endpoint viability assays in a 72-hour drug screen. Upon successful execution of this protocol the user will have access to the following raw data:

Live-cell imaging to display morphological changes and response to drug treatment at 6 concentrations per cell line over a 72-hour time span.
Endpoint cell metabolism-based viability data curves generating dose response curves for each cell line.
Untreated cell growth rates and cell viability drug response data which allows for the calculation of metrics such as IC50, AUC (area under the curve), GR50, and AOC (area over the curve).


[STEPS]
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
1. In a sterile tissue culture hood, aspirate media from t75-flask. 
 

SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
2. Perform a wash with 5 mL Phosphate Buffered Saline (PBS) (Hyclone, Cat# SH30028.02).
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
3. To detach cells from tissue culture flasks, add 3mL of trypsin (Corning, Cat# 25-052-cl) directly on to the cells. Gently tilt the flask until the surface is equally covered with trypsin then place cells into tissue culture incubator for 3 min.
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
4. After incubation, gently tap the sides of the flask to insure complete detachment of adherent cells. Return to incubator for 1 min increments if needed. 
 


SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
5. Add 3 mL of trypsin inhibitor (Thermofisher, Cat# R007100) (or equal volume to trypsin used) to deactivate the trypsin enzyme, collect all the cell suspension, and then transfer into a sterile 15mL conical tube.
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
6. Pellet cells via centrifugation at 100-300 x g, 5 min.
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
7. After centrifugation, aspirate all supernatant while being careful to not disrupt the cell pellet.
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
8. 
Uniformly resuspend the cell pellet in 1 mL of cell-specific culture media using a p1000 pipette. 
 

SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
9. Using the well-mixed cell suspension, accurately count cells via your preferred hemocytometry method to a final unit of [cells/mL].
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
10. After counting, calculate the volume of cells suspended in cell-specific culture media needed to prepare 10 mL cells at the seeding density concentration range previously determined. 
 

SECTION: Day 1: Prepare cell lines for seeding into 96-well plates via Opentrons OT-2 liquid handler
11. Transfer 10 mL of each cell suspension to columns 1-8 of a 12-channel reservoir. See schematic below.
  

SECTION: Preparing for cell seeding via Opentrons OT-2 Liquid handler
12. 
 
Instructions for tip arrangement:
Alternate between a full row of tips (for mixing in the 12-channel reservoir) in one row and then 4 tips for the corresponding quadrant where the cells will seeded. This protocol seeds the cells in the upper quadrants first, and then returns to the cells in the lower quadrants from left to right. Two tip racks will be needed for this protocol. See image below:
 
Corresponding starting deck state compatible with OT-2 seeding protocol:
  
  

SECTION: Preparing for cell seeding via Opentrons OT-2 Liquid handler
13. Transfer loaded 12-channel reservoir, pre-arranged tip racks, and two empty white walled/clear bottomed 96-well plates into the OT-2 deck and begin OT-2 procedure “Cell Seeding 8 Cell Lines.json”.
SECTION: Preparing for cell seeding via Opentrons OT-2 Liquid handler
14. Place 96-well plates into standard tissue culture incubator  .

SECTION: Day 2: Prepare drug dilutions
15. Observe cells with a microscope at 10x magnification to verify that cells are settled and adhered to the inner surface of the 96-well plate.
SECTION: Day 2: Prepare drug dilutions
16. Prepare drug at pre-determined max concentration into pre-determined optimized media conditions. For this protocol, each cell line (in the optimized media conditions) will require 2.5 µL of drug prepared at the pre-determined max concentration as well as 2.5 µL of vehicle treated media at the same percentage of the max drug concentration.

Example: If you are working with DrugX at a stock concentration of 50mM and desire a max concentration of 30µM in 2.5 mL of media use the formula:

   

where V1= Desired volume of DrugX at stock concentration to be added into optimized media conditions, C1= Concentration [µM] of DrugX at stock concentration, C2=Desired final concentration of DrugX (30 micromolar (µM)), & V2= Desired final volume of new cell preparation (2.5 mL). For the vehicle preparation, add equal volume as V1 of vehicle into media with optimized conditions.
 

SECTION: Day 2: Prepare drug dilutions
17. In a sterile 24-well plate transfer 2.266 mL of DrugX at max concentration in column 1 for each cell line using the cell-specific media conditions. Similarly, transfer 2.266 mL of vehicle treated media into column 6. In the center columns 2-5, add 1.334 mL of untreated media at optimized conditions for the corresponding cell lines (See plate map above). There should be one 24-well plate per white opaque 96-well plate (seeded the previous day).
 
Drug dilution and transfer to template clear 96-well plate schematic:
 
Attached File: “Drug Dilution and Transfer.json”
Duration: ~65 min

Corresponding starting deck state compatible with OT-2 seeding protocol:
  
   
SECTION: Day 2: Prepare drug dilutions
18. Transfer loaded 24-well plate, full tip racks, an empty standard clear 96-well plates into the OT-2 deck and begin OT-2 procedure “Drug Dilution and Transfer.json”.
SECTION: Day 2: Prepare drug dilutions
19. After drug dilution and transfer into the template standard clear 96-well plate (~43 min), the protocol pauses to allow the user to add the first 96-well plate at this step. Add plate then resume protocol.

SECTION: Day 2: Prepare drug dilutions
20. After the first plate is completed, return treated white 96-well plate into an incubator, and repeat the drug dilution and treatment procedure with the second plate (~65 min).
SECTION: Day 2: Prepare drug dilutions
21. Afterwards, second plate into the incubator and proceed to the next step.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
22. At this stage, the plate can be transferred to the preferred live-cell imaging platform. This protocol uses the IncuCyte Zoom platform. The plate will remain in the imaging platform for 4320 min .
 

SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23. 
 
Begin IncuCyte Zoom set up
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.1. Open IncuCyte Zoom software on computer desktop.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.2. Connect to device.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.3. Under the “Task List” panel on the left-hand side, select “Schedule Scans”.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.4. Click one of the “Empty” slots on the live representative plate map for the hardware then click “Add Vessel”.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.5. Once prompted, search from the vessel (96-well plate) by catalog number. This protocol features the Greiner bio one cell culture microplate (#655098). Once selected proceed to setting up experimental parameters:
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.6. On the bottom panel on the left-hand side, select “Edit Scan Pattern” and select all wells and set the scan pattern to 4 images/well. Save this scan pattern.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.7. In the Channel Selection section in the center, click “Phase” (no colored acquisition channel is need for this protocol).
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.8. In the “Scan Mode” section in on the top-right side, toggle to the scan pattern that was previously created and saved.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.9. In the “Analysis Job Setup” section on the right-hand side, toggle the “Job Type” and select “Basic Analyzer.” Toggle the “Processing Definition” tab and select a pre-determined processing definition with masks optimized for your specific cell line. (If this hasn’t been created, the “DEMO Phase” processing definition can be used.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.10. Click the “Properties” tab and label the plate as desired.
SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
23.11. Click “Apply” on the bottom right corner to save changes and register the plate to the IncuCyte Zoom hardware.
 

SECTION: Day 2: Verify cell attachment to plates after drug treatments and begin IncuCyte ZOOM live-cell imaging
24. Transfer the 96-well plates into the same IncuCyte Zoom slot selected during the software setup and begin real-time image capture.
 

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
25. After 72-hours have passed, end the experiment on the live-cell imagining platform software then remove the 96-well plate and allow and allow it to equilibrate to Room temperature ~ 20 min. 
 

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26. 
 
End IncuCyte Zoom Experiment:
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.1. Open IncuCyte Zoom software on computer desktop.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.2. Connect to device.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.3. Under the “Task List” panel on the left-hand side, select “Schedule Scans”.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.4. Click on the slot housing the plate being tested on the live representative plate map for the hardware then click “Remove Vessel”.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.5. Click “Apply” on the bottom right corner to save changes.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
26.6. Manually remove the corresponding 96-well plate to the IncuCyte Zoom hardware.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
27. Cover the bottom of the 96-well plate with white opaque lab tape.
 
See example below:
       

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
28. Transfer 10 mL (per plate) of the thawed CellTiter-Glo reaction reagent into a standard 25-mL reagent reservoir.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
29. Use an 8-channel p200 multi-channel pipette (or comparable multi-channel pipette) to transfer 100 µL of CellTiter-Glo reaction reagent into each well of the 96-well plate.

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
30. Leave the plate cover off and transfer into a luminometer compatible with CellTiter-glo. Be mindful of the plate orientation and alignment to insure proper placement into the device. See example below:
       

 

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
31. Recommended parameters for the Luminometer are as follows:
Shake in an orbital shaker for 2 min at 500 cycles/minute with a 1mm shaking diameter (cell lysis).
Incubate for 10 min in a dark environment (reaction).
Read luminescence of each well at an integration of 400ms (data acquisition).
 

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
32. Export data and remove plate from luminometer.