Add Live, Dead and Total Dyes. Overnight Variable 30 min. 10 min 5 min. 30 min

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1 Cell Viability Analysis using Calcein AM, Propidium Iodide and Hoechst Application Description Celigo Application Plate Type Major Steps Cell viability analysis using Calcein AM, Propidium Iodide and Hoechst on A549 cells. This protocol describes the analysis of cell viability on A549 cells using Calcein AM as a measure of enzymatic activity in live cells, Propidium Iodide as a measure of dead cells with compromised membrane integrity and Hoechst as a marker for all nucleated cells. The application reports the counts and percentages of live, dead and total numbers of cells. Cell Viability (Live + Dead + Total). Greiner cat# black wall clear bottom 96- well Plate. Seed cells Treat with drug Add Live, Dead and Total Dyes Image on Celigo Analyze cell images Produce graphs and data Overnight Variable 30 min. 10 min 5 min. 30 min Day 1 Day 2 Experimental Setup Materials: A549 Cells A549 growth media: EMEM + 10% FBS + 1% NEAA + 1% GlutaMax Calcein AM, 20 x 50 µg (Life Technologies, Cat# C3100MP) Propidium Iodide, 1 mg/ml solution in water (Life Technologies, Cat# P3566) Hoechst 33342, 10 mg ml solution in water (Life Technologies, Cat# H3570) DMSO Greiner 96- well plate Cat# Protocol: 1. Reconstitute Calcein AM by adding 50.2 μl of DMSO in one of the 50 µg vial of lyophilized Calcein AM. This will results in a 1 mm Calcein AM solution. 2. Seed cells/well in 96- well plate in a final volume of 100 μl/well. 3. Incubate cells overnight at 5% CO2, 37 C. Celigo Application Protocol Cell Viability Analysis 1

2 4. In the example protocol provided here, we use hydrogen peroxide to induce cell death. Below is the plate map for hydrogen peroxide concentration. Concentration should be adjusted for any other drug. Skip to paragraph 6 if using a different drug or plate map. Maximum Concentration (mm) Control (mm) A B C D E F G H Concentration Response Curve (mm) 5. The appropriate well concentrations were obtained by preparing a 2x concentration of the dilution curve as 100 μl of drug will be added to 100 μl of media in the wells. Dilution steps were obtained as follow: a. Prepare a 1M solution of hydrogen peroxide by adding 500 μl of 3% hydrogen peroxide to 4.4 ml of water. b. Prepare a 100 mm solution of hydrogen peroxide by adding 400 μl from the previous step to 3.6 ml of culture media. c. Using a series of 12 tubes, prepare a serial dilution by diluting hydrogen peroxide in media according to the following table. For 2x Conc (mm) From tube (mm) Add (µl) To Media (µl) Dilution Factor Total Vol. (µl) Final well Conc (mm) plate column Drug compound treatment a. Add 100 μl of compound at 2x desired final concentration. b. After desired treatment time (4 hours for hydrogen peroxide), wash wells with media twice and leave 100 μl volume in wells. Celigo Application Protocol Cell Viability Analysis 2

3 7. Prepare Calcein AM, Propidium Iodide and Hoechst mixed dye solution. a. 12 ml of mixed dyes solution is required for staining all the wells of a 96- well plate. Adjust accordingly if not all wells are stained. b. Use the following dilution table to prepare the mixed dye solution Dye Volume to add Concentration Stock Recommended Recommended to 12 ml range for other concentration concentration dilution factor media cell types Calcein AM 1 mm 1 μm 1000x 12 μl μm Propidium Iodide 1 mg/ml 2 μg/ml 500x 24 μl μg/ml Hoechst mg/ml 5 μg/ml 2000x 6 μl 1 10 μg/ml c. Remove media from all wells and add 100 μl of mixed dyes solution d. Incubate cells for 30 min at 5% CO2, 37 C. e. Wash wells with media twice and leave 100 μl volume in wells. f. As cells are live in the wells, the plate should be imaged immediately on the Celigo. Celigo Setup Start Tab: 1. Create a new scan and name file appropriately. 2. If experimental settings had been previously optimized then select these to be used. Scan Tab: 1. Select Celigo Application: Cell Viability (live + Dead + Total). 2. Setup Live Channel a. Select Green illumination for Calcein AM stain. b. Exposure time should be around 10,000 μs (Gain 0). 3. Setup Dead Channel a. Select Red illumination for Propidium Iodide stain. b. Exposure time should be around 40,000 μs (Gain 0). 4. Setup Total Channel a. Select Blue illumination for Hoechst stain. b. Exposure time should be around 100,000 μs (Gain 0). Celigo Application Protocol Cell Viability Analysis 3

4 Live Dead Total 5. Register Hardware autofocus using the Blue / Hoechst channel. 6. Switch to Green / Calcein AM channel, Find focus and Set Offset. 7. Switch to Red / Propidium Iodide channel, Find focus and Set Offset. 8. Select wells to acquire and Start Scan. Analyze Tab: The Celigo Cell Viability application segments all three fluorescent channels. Fluorescent objects will be identified in each channel. Green and Red objects (for live and dead cells) will be counted only if they are super- imposed with a blue fluorescent object (for nuclear stain). Therefore, segmented green and red objects (such as fluorescent debris) not associated with a blue nucleus will be rejected from the analysis. 1. Select Well Mask and Automatic to exclude the outer part of the well. 2. Select Fluorescence algorithm for all 3 channels. 3. Adjust the Intensity Threshold to ensure proper identification of the nucleus. Default value of 4 with High Precision works well for most fluorescent stains. Values of 2 to 3 can be used for dimmer samples. 4. Keep the Cell Diameter between 8 and Select Separate Touching Objects in the Total channel to ensure proper separation of nuclei in close proximity. Live Dead Total Celigo Application Protocol Cell Viability Analysis 4

5 6. Expected Segmentation: a. Use the Image Display and Graphic Overlay controls to visualize segmentation. b. Typical images and fluorescent objects identification should look as follow: Live Dead Total Segmentation Images Celigo Application Protocol Cell Viability Analysis 5

6 Live + Dead + Total Segmentation Overlays Live cell with green overlay super- imposing a blue nucleus overlay Dead cell with red overlay super- imposing a blue nucleus overlay Green overlay dismissed from analysis because not super- imposing a blue nucleus overlay Red overlay dismissed from analysis because not super- imposing a blue nucleus overlay 7. Select wells to analyze and Start Analyze. Gate Tab: The Cell viability application does not use gating. Instead, quantification of live and dead cells is pre- defined in the application and available directly in the Result Tab. Result Tab: 1. Typical Results for the percentage of live cells. Control No Treatment Hydrogen Peroxide (12 mm) Hydrogen Peroxide Conc. Response Celigo Application Protocol Cell Viability Analysis 6

7 Data Analysis The example provided in this protocol uses A549 cells treated with a concentration response curve of hydrogen peroxide, an inducer of cell death. As hydrogen peroxide concentration increases, we expect to a decrease of the percentage of live cells and an increase percentage of dead cells. Data Export 1. Export the data by selecting Export Well- Level Data 2. Save the.csv file to your desktop. 3. Open the file with Microsoft Excel. 4. The data is organized according to the plate map for each of live and dead cells classes. % Live A 86.92% 90.05% 90.93% 94.20% 92.86% 91.73% 0.10% 0.02% 0.04% 0.07% 0.03% 0.00% B 95.30% 97.64% 95.07% 95.69% 96.80% 96.68% 0.38% 0.27% 0.29% 0.13% 0.23% 0.04% C 95.61% 96.69% 95.89% 96.54% 97.51% 97.42% 0.32% 0.60% 0.48% 0.27% 0.28% 0.12% D 96.62% 97.89% 96.10% 98.13% 97.79% 97.59% 0.23% 0.19% 0.32% 0.18% 0.12% 0.07% E 0.02% 0.95% 2.21% 4.31% 8.40% 80.73% 97.26% 97.94% 97.27% 92.94% 93.37% 95.46% F 0.04% 0.57% 1.75% 3.77% 8.54% 82.48% 97.52% 97.70% 96.77% 90.97% 94.21% 95.11% G 0.08% 0.59% 1.58% 7.89% 72.01% 96.78% 97.85% 97.52% 84.33% 79.70% 86.01% 94.15% H 0.01% 0.17% 0.90% 1.93% 15.78% 54.26% 97.22% 97.73% 87.77% 86.77% 83.21% 95.14% % Dead A 1.05% 1.05% 1.19% 1.47% 1.57% 1.44% 81.08% 80.68% 79.42% 79.08% 80.68% 78.34% B 1.11% 1.18% 1.33% 1.33% 1.10% 1.18% 74.68% 76.62% 77.77% 74.93% 77.09% 78.48% C 1.01% 0.89% 0.84% 1.20% 1.36% 1.50% 71.45% 72.90% 70.81% 72.09% 75.67% 79.03% D 0.97% 0.92% 1.14% 1.06% 1.19% 0.93% 67.78% 72.30% 72.66% 71.54% 75.25% 80.36% E 80.55% 81.84% 87.48% 88.49% 86.34% 17.13% 1.61% 2.19% 1.83% 1.49% 1.17% 1.13% F 78.30% 81.28% 86.58% 89.36% 86.27% 15.85% 1.95% 1.86% 1.16% 0.94% 1.03% 0.79% G 84.44% 88.56% 91.97% 86.97% 22.62% 1.39% 1.43% 1.64% 1.03% 0.76% 0.94% 0.88% H 82.50% 86.32% 87.84% 92.38% 78.13% 39.31% 2.90% 2.61% 1.57% 0.77% 1.10% 1.10% Graphing 1. Generate a Scatter plot using Microsoft Excel using the drug concentration for the X- axis and 2 series for the live and dead cells. In this example, the average of 4 data points were plotted. 2. You should expect the following results: Live and Dead percentages of A549 cells treated with Hydrogen Peroxide 100% Percentage of Total 80% 60% 40% 20% 0% % Live % Dead 1 10 Hydrogen Peroxide (mm) Celigo Application Protocol Cell Viability Analysis 7

8 Variations of the Celigo Cell Viability application Three other variants of the Cell Viability application exist in the Celigo software. All three of them are 2 channels applications representing a subset of the 3 channel protocol describe here. In this variants, the settings for the Live, Dead and Total channels remain the identical to the 3 channels application. Additionally, the Total channel can be changed from using a blue nuclear dye to brightfield where the cells can also be counted. The following table provides details for each application. Cell Viability Application Live channel Dead Channel Total Channel Application outputs Live + Dead + Total Green Red Blue or Brightfield % Live, % Dead, Total counts Live + Total Green Not Available Blue or Brightfield % Live, Total counts Dead + Total Not Available Red Blue or Brightfield % Dead, Total counts Live + Dead Green Red Not Available % Live, % Dead, Total counts Note: Additional outputs are reported by the Cell Viability application. Refer to Cell Viability user guide for a complete list of outputs. Reference Chen HY, Yang YM, Stevens BM, Noble M. EMBO Mol. Med (2013). Inhibition of redox/fyn/c- Cbl pathway function by Cdc42 controls tumour initiation capacity and tamoxifen sensitivity in basal- like breast cancer cells. Celigo Application Protocol Cell Viability Analysis 8