TITLE: 96-well plate cell growth optimization for integrated live-cell and endpoint viability drug screening assay
AUTHORS: Rolando DZ Lyles, M Julia Martinez, Benjamin Sherman, Kerry Burnstein

[DESCRIPTION]
This protocol details the cell growth optimization for integrated live-cell and endpoint viablility drug screening assay on a 96-well plate.

[BEFORE_START]
Before starting:

Determine the cell line to be optimized and expand cell line to ~50-60% confluence in any standard tissue t75-Flask or 15cm tissue culture dish.
Define cell seeding density range to be tested. This protocol uses 4 seeding densities. Always set initial seeding density to the cell line-specific lab standard or literature recommended seeding density for 96-well plate assays. Then set a range using 50% increments (one below and two above) of initial seeding density.
 

Determine desired cell culture media conditions to be tested. This protocol 2 media conditions with varying serum concentrations. As a reference point, this protocol suggests preparing cell culture media with cell specific recommended serum concentrations and preparing cell culture with between 50-80% less serum to regulate cell growth rates.

 


[GUIDELINES]

To accurately integrate live-cell imaging and endpoint viability, the user must define conditions which promote cell growth, prevent over-confluence, and fall within the detectable linear range of the endpoint cell viability assay. This protocol is designed to identify the optimal cell seeding density and media conditions to be used for integrated live-cell imaging and endpoint metabolic ATP viability assays for 72-hour drug screens using 96-well plates.
 

This protocol is designed to identify optimized seeding conditions for a single cell line so that it is compatible for sequential live-cell imaging and endpoint viability assays in a 72-hour drug screen. Successfully optimized seeding densities and conditions meet the following basic criteria:

Live-cell imaging displays healthy cells which are growing throughout 72-hours.
Cells aren’t overconfluent at 72-hour endpoint. (We recommend a final confluence ≤ 60%, however, some slow growing cell lines may need to be seeded more densely and, in these cases, avoid conditions where cells are growing in clumps at experimental endpoint).
The range of seeding densities (at each condition) fall within the detectable linear range of the endpoint viability assay. 

The user may find a range of options that fit these criteria. In these cases, it is to the user’s discretion to select the conditions that align best with their study objective. 

Tips for data analysis:

Track confluence throughout live-cell imaging steps- at endpoint export confluence data for all wells as an average of each of the images. This will allow you to calculate growth rate as well as final average endpoint confluence.
To determine if seeding densities are within the detectable linear range of the endpoint viability assay, Create an X-Y scatterplot where raw luminescence values averaged for each replicate is graphed at each seeding density. Perform a linear regression or linear curve fit to calculate the R2 (a value which denotes how well the data “fits” the linear curve). Note: R2 values range from 0-1 where ≥ 0.9 is considered a good fit.
If users are planning to optimize multiple cell lines for comparative drug screens, then it is recommended to select conditions which allow the most similarities across cell lines. I.e. if one cell line is only within an optimized range when 2% FBS is used but another is in an optimized ranged at either 2% or 5% FBS, proceed with both cell lines using 2% FBS to limit variables.


[STEPS]
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates for optimization
1. In a sterile tissue culture hood, aspirate media from t75-flask.
SECTION: Day 1: Prepare cell lines for seeding into 96-well plates for optimization
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 for optimization
3. To detach cells from tissue culture flasks, add 3 mL 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 for optimization
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 for optimization
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 for optimization
6. Pellet cells via centrifugation at 100-300 x g, 5 min.
 

SECTION: Day 1: Prepare cell lines for seeding into 96-well plates for optimization
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 for optimization
8. Uniformly resuspend the cell pellet in 1mL of SERUM FREE media using a p1000 pipette. 
 
*Then add an additional 1 mL of SERUM FREE media and slowly vortex to uniformly mix.

SECTION: Day 1: Prepare cell lines for seeding into 96-well plates for optimization
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: Seeding cells into 96-well plates for optimization
10. 
 
After counting, calculate the volume of cells suspended in SERUM FREE media needed to prepare 12 mL at the adjusted seeding density concentration range previously determined.
 


SECTION: Day 1: Seeding cells into 96-well plates for optimization
11. Using the two media conditions prepared between steps 6 & 7, prepare 3 mL of each seeding density/ media condition combination in separate sterile 15 mL conical tubes.

Example table:
     Cell   Line      Seeding   Density      Media   Condition      Final   Volume        Cell X      500 cells/well      Condition 1      3mL        Cell X      1000   cells/well      Condition   1      3mL        Cell X      1500 cells/well      Condition 1      3mL        Cell X      2000   cells/well      Condition   1      3mL        Cell X      500 cells/well      Condition 2      3mL        Cell X      1000   cells/well      Condition   2      3mL        Cell X      1500 cells/well      Condition 2      3mL        Cell X      2000   cells/well      Condition   2      3mL   
 
SECTION: Day 1: Seeding cells into 96-well plates for optimization
12. One at a time, gently vortex each tube and transfer into a standard sterile standard 25 mL reagent reservoir (VWR, Cat# 89094-662). Then using an 8-channel p200 multi-channel pipette, transfer the cell suspensions into a cell culture 96-well microplate with white opaque walls and a clear bottom (Sigma Aldrich, M0437-32E) at their proper locations as specified by the above plate map.
 

SECTION: Day 1: Seeding cells into 96-well plates for optimization
13. Place 96-well plate into standard tissue culture incubator 5 min.

SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
14. 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. Verify that there is a reasonable difference in expected seeding densities.
 

SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
15. 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 (the duration of the drug screens in the integrated live-cell and endpoint viability drug screen protocol).

SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16. The instructions below apply exclusively to the IncuCyte Zoom platform which this protocol features. 
 
Begin IncuCyte Zoom set up:
SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16.1. Open IncuCyte Zoom software on computer desktop.
SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16.2. Connect to device.
SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16.3. Under the “Task List” panel on the left-hand side, select “Schedule Scans”.
SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16.4. 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 and begin IncuCyte ZOOM live-cell imaging
16.5. 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 and begin IncuCyte ZOOM live-cell imaging
16.6. 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 and begin IncuCyte ZOOM live-cell imaging
16.7. 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 and begin IncuCyte ZOOM live-cell imaging
16.8. 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 and begin IncuCyte ZOOM live-cell imaging
16.9. 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 and begin IncuCyte ZOOM live-cell imaging
16.10. Click the “Properties” tab and label the plate as desired.
SECTION: Day 2: Verify cell attachment to plates and begin IncuCyte ZOOM live-cell imaging
16.11. 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 and begin IncuCyte ZOOM live-cell imaging
17. Transfer the 96-well plate 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
18. 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
19. 
 
End IncuCyte Zoom Experiment: 
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
19.1. Open IncuCyte Zoom software on computer desktop.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
19.2. Connect to device.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
19.3. 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
19.4. 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
19.5. 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
19.6. 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
20. 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
21. 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
22. 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
23. Leave the plate cover off and transfer into a luminometer compatible with CellTiter-glo. 
 

See example below:
  
  
 

SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
25. Export data and remove plate from luminometer.
SECTION: Day 5: End live-cell imaging procedure and perform CellTiter-glo endpoint viability assay
24. 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).