Celigo Assays.

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1 Celigo Assays April

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3 Nexcelom's team of Field Applications Scientists, R&D Specialists and Product Managers are in frequent contact with researchers in the field, developing new experiments that can be performed on the Celigo image cytometer. We have taken this information and generated three unique types of documents: Celigo Assays: In these red-colored documents, we summarize in 1-2 pages a basic overview of how certain assays would be performed on the Celigo. We include any stains needed, the imaging channels used on the Celigo, and a truncated review of methods and results generated from the assay. These are quick snapshots, providing proof of concept of specific assays that have been performed successfully on the Celigo. Celigo Demonstration Experiments: In these green-colored documents, we outline in detail specific experiments that have been conducted on the Celigo. In these demonstration experiments we highlight the plate set up, assay protocol, detailed results generated by the Celigo, as well as a conclusion and summary of the outcome of the experiment. Celigo Application Protocols: In these blue-colored documents, we provide a step-by-step guide on how to prepare, perform and analyze specific assays using the Celigo image cytometer. These are designed to comprehensively walk users through every step of the experiment: from prep to data analysis. Our team continues to create new Celigo Assays, Demonstration Experiments and Applications Protocols documents. For the most current materials, please visit our website or reach out to your local area manager or applications scientist. The chart on the next page depicts the Celigo Assays, Celigo Demo Experiments and Celigo Applications Protocols that currently exist for different applications and assays. If you are interested in viewing any of the Celigo Assays or Celigo Demo Experiment documents, they are individually readily available on for download. Simply type the name of the assay into the search bar on the homepage. We have also created an ebook version of all our Celigo Experiment Demonstrations and all our Celigo Application Protocols. 3

4 Celigo Assays Celigo Demos Celigo Application Protocols Immuno-Oncology 01_0001 NK Cell Mediated Cytotoxicity w/ Calcein AM X X X 01_0002 NK Cell Mediated ADCC w/calcein AM X 01_0003 Neutrophil Mediated ADCC w/calcein AM X 01_0004 CDC w/calcein AM X X 01_0005 ADC Mediated Cytotoxicity w/bf X X 01_0008 CIK Mediated ADCC w/cfse & PI X 01_0009 CAR T T Cell Mediated Cytotoxicity w/cf SE & PI X 01_0010 NK92 Cell Mediated ADCC w/calcein AM X X X 01_0011 PBMC Mediated Cytotoxicity using Calcein AM X X X Fluorescent Assays 02_0001 Cell Cycle w/dapi & BrdU X X 02_0002 Viability w/calcein AM, Hoechest & PI X X 02_0003 Endpoint Apoptosis w/caspase 3/7 & Hoechst X X X 02_0004 Cell Cycle w/edu & DAPI X 02_0005 Endpoint Viability w/draq7 & Hoechst X X X 02_0006 Kinetic Viability w/draq7 X X X 02_0007 HT Viability using AO/PI X X X 02_0008 HT Viability using AO/PI & BF X 02_0009 p53 & phosphor-p53 FL Marker Analysis X X 02_0010 Endpoint Viability w/pi & Hoechst X X X 02_0011 Kinetic Viability w/pi X X X 02_0012 Kinetic Apoptosis w/caspase 3/7 X X X 02_0013 Cell Cycle PI Analysis using A375 cells X 02_0014 HPC proliferation measurement using Ki-67 cellular marker X X X 02_0015 Antibody-Dependent Receptor Internalization Assay X 02_0016 Count and measure cell infectivity of microcarriers X 02_0017 Antibody Cell Surface Binding Detection X X 02_0018 GFP Transfection Efficiency Measurement X 02_0019 Antibody Dependent Drug Uptake Assay X 02_0020 Kinetic phrodo Antibody Internalization using Confluence X X 02_0021 Kinetic Antibody Internalization using phrodo and Bright Field X X 02_0022 Endpoint Antibody Internalization using phrodo and Hoechst X X 3D Models 03_0001 Label Free Tumor Spheroid Growth Inhibition X X 03_0002 3D Multicellular tumor sphere (MCTS) invasion screen X X X 03_0003 3D MCTS Size and Morphology Determination Assay X 03_0004 3D MCTS growth inhibition screening X X X 03_0005 3D MCTS endpoint apoptosis screening X X X 03_0006 Counting of Patient Derived idc Organoids X 03_0007 3D MCTS Endpoint Viability Screening Assay X X X 03_0008 3D MCTS Kinetic PI Assay X X X Migration/Invasion 04_0001 Transwell Migration w/dapi X 04_0002 Scratch Wound Healing Assay using Direct Cell Counting X X 4

5 04_0003 Scratch Wound Healing Assay using Confluence X X 04_0004 2D Cell Migration Assay with Oris Platypus Plate X X X Cell Counting 05_0001 Label-Free adherent cell proliferation using confluence X X 05_0002 Label-Free suspension cell proliferation by direct cell counting X X Bright Field Cell Line Development 07_0001 Single Cell Detection of FACS-sorted cells X ipsc Reprogramming 5

6 Assay Name: NK cell-mediated cytotoxicity using calcein AM Assay ID: Celigo_01_0001 Description: Measure NK cell direct killing by counting total live tumor cells Stains: calcein AM (green total live cells) Imaging channels: Bright field and green Image analysis algorithm: Celigo software Target Culture and collect K562 and IMR32 Target cells and stain with calcein AM (Nexcelom, Cat# CS1-0119) 2. Seed the Target cells in the wells of a 96-well microplate 3. Add the NK effector cells at E:T ratios of 10:1, 5:1, 2.5:1, 1.3:1, 0.6:1, and 0.3:1 4. Co-culture the K562 or IMR32 cells with cultured NK Effector cells for 4 hours and observe the NK cellmediated cytotoxicity 5. Use Celigo and capture images hourly and analyze the total number of live Target cells over time 6. Use the equation below to calculate cytotoxicity a. % Cytotoxicity = 1 -./ :;<9;= -./ >?@9:?A E:T ratio-dependent NK cel-mediated cytotoxicity % Cytotoxicity 100% 80% 60% 40% 20% 0% 10:1 5:1 2.5:1 1.3:1 0.6:1 0.3:1 E:T Ratio The calcein AM fluorescent images showed a reduction in Target cell number as the E:T ratios decreased The % cytotoxicity is plotted to show the effect of E:T ratios on Target cell cytotoxicity Celigo Assay NK cell-mediated cytotoxicity using calcein AM Rev B

7 Time-dependent NK cell-mediated cytotoxicity % Cytotoxicity 100% 80% 60% 40% 20% 0% 10:1 5:1 2.5:1 1.3:1 0.6:1 0.3: Incubation Time (Hour) As time increased, the number of calcein AM-stained Target cells decreased Different E:T ratios showed different NK cell-mediated cytotoxicity effect on the Target cells Celigo Assay NK cell-mediated cytotoxicity using calcein AM Rev B

8 Assay Name: NK cell-mediated ADCC using calcein AM Assay ID: Celigo_01_0002 Description: Measure NK cell-mediated ADCC by counting total calcein AM-positive, live tumor cells Stains: calcein AM (green total live cells) Imaging channels: Bright field and green Image analysis algorithm: Celigo software Target Culture and collect Target cells and stain with calcein AM (Nexcelom, Cat# CS1-0119) 2. Seed the Target cells in the wells of 96-well microplate 3. Add the NK Effector cells and different concentrations of antibodies 4. Co-culture the Target cells with cultured NK Effector cells with antibodies for 4 hours and observe the NK cell mediated ADCC 5. Use Celigo to capture images hourly and analyze the total number of calcein AM-positive live Target cells over time 6. Use the 2 equations to calculate cytotoxicity a. Normalized to t = 0 i. % Cytotoxicity normalized to time = 1 -./ JK -./ JL b. Normalized to spontaneous release i. % Cytotoxicity = % cytotoxicity sample % cytotoxicity (spont) NK cell-mediated ADCC bright field and fluorescent overlay images showed increased cytotoxicity over time The number of calcein AM-positive Target cells decreased as incubation time increased with NK cells The morphology of NK cells and Target cells are visually different It also showed that the NK cells cluster around the Target cells to induce cytotoxicity Celigo Assay NK cell-mediated ADCC using calcein AM Rev B

9 NK cell-mediated ADCC time- and dose-dependent graphs % Cytotoxicity 40.0% 35.0% 30.0% 25.0% 20.0% 15.0% 10.0% 5.0% 0.0% NK cell-mediated ADCC time-dependent results Time (min) Ab 1 Ab 2 Ab 3 Ab 4 Ab 5 Ab 6 Ab 7 In the time-dependent graph, different [antibody] showed different levels of % cytotoxicity over time % Cytotoxicity 30% 25% 20% 15% 10% 5% 0% -5% NK cell-mediated ADCC dose-dependent results [Antibody] Time 1 Time 2 Time 3 Time 4 In dose-dependent graph, the dose response also showed different levels of % cytotoxicity as [antibody] increased Celigo Assay NK cell-mediated ADCC using calcein AM Rev B

10 Assay Name: Neutrophil-mediated ADCC using calcein AM Assay ID: Celigo_01_0003 Description: Measure neutrophil ADCC by counting total calcein AM-positive, live tumor cells Stains: calcein AM (green total live cells) Imaging channels: Bright field and green Image analysis algorithm: Celigo software Target Culture and collect Target cells and stain with calcein AM (Nexcelom, Cat# CS1-0119) 8. Seed the Target cells in the wells of 96-well microplate 9. Add the neutrophil Effector cells and different concentrations of antibodies 10. Co-culture the Target cells with cultured neutrophil Effector cells with antibodies for 4 hours and observe the neutrophil mediated ADCC 11. Use Celigo to capture images hourly and analyze the total number of calcein AM-positive live Target cells over time 12. Use the 2 equations to calculate cytotoxicity a. Normalized to t = 0 i. % Cytotoxicity normalized to time = 1 -./ JK -./ JL b. Normalized to spontaneous release i. % Cytotoxicity = % cytotoxicity sample % cytotoxicity (spont) Neutrophil-mediated ADCC time- and dose-dependent results % Cytotoxicity 50% 40% 30% 20% 10% Neutrophil-mediated ADCC timedependent results Control 2 Control 1 Ab 3 Ab 2 Ab 1 % Cytotoxicity 45% 35% 25% 15% 5% Neutrophil-mediated ADCC dosedependent results Control Ab 0% -5% Time (Hour) [Antibody] As time increased, the Target cell cytotoxicity increased for the positive antibody, while the control did not show significant change Celigo Assay Neutrophil-mediated ADCC using calcein AM Rev B

11 The dose response showed antibodies induced a high neutrophil-mediated ADCC effect to the Target cells Neutrophil-mediated ADCC bright field and fluorescent overlay images Ab Control As time increased, the number of calcein AM-stained Target cells decreased In the control antibody, the neutrophils did not induce cytotoxicity, where the calcein AM-positive cells remained bright green It is clear in the bright field and fluorescent overlay images that the neutrophils elongated and surrounded the Target cells, which induced cytotoxicity (shown with the red arrows) Celigo Assay Neutrophil-mediated ADCC using calcein AM Rev B

12 Assay Name: Complement-dependent cytotoxicity using calcein AM Assay ID: Celigo_01_0004 Description: Measure complement-dependent cytotoxicity (CDC) by counting total calcein AM-positive, live tumor cells Stains: calcein AM (green total live cells) Imaging channels: Bright field and green Image analysis algorithm: Celigo software Target Culture and collect Target cells and stain with calcein AM (Nexcelom, Cat# CS Seed the Target cells in the wells of 96-well microplate 15. Add different concentrations of antibodies 16. Add serum 17. Use Celigo and capture images hourly and analyze the total number of live Target cells over time 18. Use the 2 equations to calculate cytotoxicity a. Normalized to t = 0 i. % Cytotoxicity normalized to time = 1 -./ JK -./ JL b. Normalized to spontaneous release i. % Cytotoxicity = % cytotoxicity sample % cytotoxicity (spont) CDC time- and dose-dependent results CDC Time-Dependent Results CDC Dose-Dependent Results % Cytotoxicity 100% 80% 60% 40% 20% 0% Control Ab 1 Ab 2 Ab 3 Ab 4 Ab 5 Ab 6 % Cytotoxicity 100% 80% 60% 40% 20% 0% Control Ab Time (min) [Antibody] As time increased, the Target cell cytotoxicity increased for the positive antibody, while the control did not show significant change The endpoint dose response showed positive antibody-induced high CDC effect to the Target cells Celigo Assay Complement-dependent cytotoxicity using calcein AM Rev B

13 CDC bright field and fluorescent overlay images As time and antibody concentration increased, the number of calcein AM-stained Target cells decreased In the top three images, where high [antibody], high cytotoxicity is shown, where almost 100% of the cells lost calcein AM fluorescence (indicated with the red arrows) In the bottom three images, where low [antibody], lower cytotoxicity is shown, with numerous calcein AMpositive, live tumor cells present (indicated with the red arrows) Celigo Assay Complement-dependent cytotoxicity using calcein AM Rev B

14 Assay Name: ADC-mediated cytotoxicity using bright field Assay ID: Celigo_01_0005 Description: Measure antibody drug conjugate (ADC) mediated cytotoxicity by measuring changes in % confluence over time Stains: Label free Imaging channels: Bright field Image analysis algorithm: Celigo software Confluence 19. Culture and collect Target cells 20. Seed the Target cells in the wells of 96-well microplate 21. Add different concentrations of antibody drug conjugates (ADCs) 22. Use Celigo and capture images daily and analyze the % confluence over time 23. Use the equation to calculate cytotoxicity a. % Cytotoxicity (control) = 1 % -63Q/ :;<9;= % -63Q/71301 >?@9:?A ADC-mediated cytotoxicity bright field-filled whole well images % Confluence 100% 75% 50% 25% 0% Dose-Dependent ADC mediated cytotoxicity [Antibody] Control Positive Cell count and % confluence can be measured using Celigo Resulting cell proliferation kinetics based on % confluence can be directly plotted to observe the effect of antibodies on Target cells High [antibody], high cytotoxicity and growth inhibition are shown, where the cell area coverage or % confluence are clearly observed in the filled view of the wells Celigo Assay ADC-mediated cytotoxicity using bright field Rev B

15 ADC time-dependent bright field images The captured images showed increasing in % confluence for the control and positive ADC-mediated cytotoxicity effect on the treated Target cells ADC time-dependent growth curves generated in Celigo The time-course kinetic plots from the Celigo software showed reduction in % confluence over time The Control sample showed that at different concentrations of antibody, the growth curves remained consistent The positive antibody sample showed slowed growth at the highest antibody dosage Celigo Assay ADC-mediated cytotoxicity using bright field Rev B

16 Assay Name: CIK-mediated ADCC using CFSE and PI Assay ID: Celigo_01_0008 Description: Measure CIK-mediated ADCC by counting total CFSE positive/pi negative live tumor cells Stains: CFSE (green total cells); Propidium Iodide (red dead cells) Imaging channels: Bright field, Green, Red Image analysis algorithm: Celigo software Target Culture and collect Target cells and stain with CFSE 25. Seed the Target cells in the wells of 96-well microplate 26. Add the CIK Effector cells and titrations of antibodies 27. Co-culture the Target cells with cultured CIK Effector cells with antibodies for 24 hours and observe the CIK ADCC 28. Stain the cells in the well with PI to identify the dead cells (Nexcelom, Cat# CS1-0109) 29. Use Celigo and capture images at Target cells over time 30. Use the equation to calculate cytotoxicity a. Cytotoxicity % = R1.S T.UV R1.SWX2Y1 T.UV CIK-mediated ADCC cytotoxicity detection method Direct visualization of cell classification based on flow-like gating Celigo Assay CIK-mediated ADCC using CFSE and PI Rev B

17 Visually, number of dead Target cells (Yellow), increased as the [antibody] increased CIK-mediated ADCC dose-dependent results 100 % Cytotoxicity [Antibody] At the endpoint, a clear cytotoxicity dose response is shown in respect to [antibody] The % cytotoxicity is calculated by the equation previously shown As the [antibody] increased, the % cytotoxicity also increased Celigo Assay CIK-mediated ADCC using CFSE and PI Rev B

18 Assay Name: CAR T cell-mediated cytotoxicity using CFSE and PI Assay ID: Celigo_01_0009 Description: Measure Chimeric Antigen Receptor (CAR) T cell mediated cytotoxicity by counting total CFSE positive/pi negative live tumor cells Stains: CFSE (green total cells); PI (red dead cells) Imaging channels: Bright field, Green, Red Image analysis algorithm: Celigo software Target Culture and collect Target cells and stain with CFSE 32. Seed the Target cells in the wells of 96-well microplate 33. Add the CAR T cells at different E:T ratios 34. Co-culture the Target cells with cultured CAR T cells for 4 hours and observe the CAR T cell killing 35. Stain the cells in the well with PI to identify the dead cells (Nexcelom, Cat# CS1-0109) 36. Use Celigo and capture images at Target cells over time 37. Use the equation to calculate cytotoxicity a. % Cytotoxicity = R1.S T.UV R1.SWX2Y1 T.UV CAR T cell killing bright field and fluorescent overlay images Visually, more CAR T cells are shown in the bright field overlay at higher E:T ratio Celigo Assay CAR T cell-mediated cytotoxicity using CFSE and PI Rev B

19 CAR T cell-mediated cytotoxicity gating results The number of dead Target cells increased as the E:T ratio increased, which can be visually seen in the scatter plots The gating allowed the measurement of number of live and dead Target cells to calculate the final % cytotoxicity CAR T cell mediated cytotoxicity results % Cytotoxicity 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% Control Positive E:T Ratio At the endpoint, a clear cytotoxicity response is shown in respect to the E:T ratios The % cytotoxicity is calculated by the equation previously shown As the E:T ratio increased, the % cytotoxicity also increased Celigo Assay CAR T cell-mediated cytotoxicity using CFSE and PI Rev B

20 Assay Name: NK92 cell-mediated ADCC using calcein AM Assay ID: Celigo_01_0010 Description: Measure antibody-dependent NK92 cell-mediated cytotoxicity (ADCC) by counting live MDA-MB- 231 cancer cells over time Stains: calcein AM (green total live cells) Imaging channels: Bright field and Green Image analysis algorithm: Celigo software Target Culture and collect MDA-MB-231 Target cells and stain with calcein AM (Nexcelom, Cat# CS1-0119) 39. Seed the Target cells in the wells of a 96-well microplate 40. Add the NK92 Effector cells and titrations of antibodies 41. Co-culture the Target cells with cultured NK Effector cells with antibodies for 6 hours 42. Use Celigo and capture images hourly and analyze the total number of live Target cells over time 43. Use the 2 equations to calculate cytotoxicity a. Normalized to t = 0 i. % Cytotoxicity normalized to time = 1 -./ JK -./ JL b. Normalized to spontaneous release i. % Cytotoxicity = % cytotoxicity sample % cytotoxicity (spont) NK92 cell-mediated ADCC fluorescent and counted images Celigo Assay NK92 cell-mediated ADCC using calcein AM Rev B

21 The number of calcein AM-positive Target cells decreased as incubation time increased with NK cells NK92 cell mediated ADCC dose-dependent results at different time points The level of cytotoxicity increased as the incubation time increased The dose response also showed different level of % cytotoxicity as [antibody] increased Immune-complex formation As the antibody concentration and incubation time increased, more immune-complex form, which are clusters of immune cells surrounding the live/dead Target cells Celigo Assay NK92 cell-mediated ADCC using calcein AM Rev B

22 Assay Name: PBMC-mediated cytotoxicity using calcein AM Assay ID: Celigo_01_0011 Description: Measure PBMC-mediated cytotoxicity by counting total live K562 cells over time in the presence and absence of IL2 Stains: calcein AM (green total live cells) Imaging channels: Bright field and Green Image analysis algorithm: Celigo software Target Culture and collect K562 cells and stain with calcein AM (Nexcelom, Cat# CS1-0119) 45. Seed the K562 cells in the wells of a 96-well microplate 46. Add the PBMC Effector cells at different E:T ratios 47. Add in IL2 to half of the wells 48. Co-culture the Target cells with PBMCs in the presence and absence of IL2 for 4 hours and measure the antibody-dependent PBMC-mediated cytotoxicity level 49. Use Celigo and capture images hourly and analyze the total number of live Target cells over time 50. Use the 2 equations to calculate cytotoxicity a. Normalized to t = 0 i. % Cytotoxicity normalized to time = 1 -./ JK -./ JL b. Normalized to spontaneous release i. % Cytotoxicity = % cytotoxicity sample % cytotoxicity (spont) PBMC-mediated cytotoxicity results of co-culture K562 and PBMC in the presence and absence of IL2 The number of calcein AM-positive Target cells increased as the E:T ratio decreased with PBMCs It also showed that Target cells with the presence of IL2 induced higher cytotoxicity Celigo Assay PBMC-mediated cytotoxicity using calcein AM Rev B

23 % Lysis 80% 70% 60% 50% 40% 30% 20% 10% 0% -10% Dose Response at T = 4 hour E:T Ratios Donor 1 - IL2 Donor 2 - IL2 Donor 1 + IL2 Donor 2 + IL2 Target samples with IL2 showed much higher cytotoxicity in comparison to samples without IL2 PBMC-mediated cytotoxicity time-dependent fluorescent images and results of co-culture K562 and PBMC E:T - 50:1 % Lysis 80% 70% 60% 50% 40% 30% 20% 10% 0% -10% Time (hour) Donor 1 - IL2 Donor 1 + IL2 Donor 2 - IL2 Donor 2 + IL2 Target Only PBMC-induced cytotoxicity increased over time for both +IL2 and -IL2 Celigo Assay PBMC-mediated cytotoxicity using calcein AM Rev B

24 Assay Name: Cell cycle analysis using DAPI and BrdU Assay ID: Celigo_02_0001 Description: Analysis of cell cycle using DAPI and BrdU incorporation on A549 cells Stains: Alexa Fluor 488 goat anti-mouse antibody and DAPI Imaging channels: Green and Blue Image analysis algorithm: Celigo software Expression Analysis: Target 1 + Mask 1. Seed cells in 96-well plate and incubate cells overnight 2. Add 100 μl of compound at 2x desired final concentration (e.g. Aphidicolin, an inhibitor of DNA synthesis) 3. BrdU incorporation step, 100 μm BrdU and incubate for 2 hours 4. Fixation and DNA denaturation step, 200 μl/well of Ethanol/Glycine solution for 30 minutes 5. Antibody labeling steps: a. 50 μl Anti-BrdU mouse monoclonal antibody and incubate for 2 hr b. 50 μl Anti-BrdU Alexa Fluor 488 goat anti-mouse antibody and incubate for 1 hr and wash 6. Add 100 μl DAPI mix to each well and incubate for 30 min 7. Image and gate on the Celigo Gating interface displaying the horseshoe plot and cells are color-coded dependent on the phase of the cell cycle they are in. Gating Interface Plot Cell Segmentation BrdU integrated intensities vs DAPI integrated intensities are plotted to create the horseshoe shape in the above left image. S-phase cells are identified in green, G0/G1 cells in purple and G2/M in blue in a control well in the above right image. Celigo Assay Cell cycle analysis using BrdU and DAPI Rev A

25 Aphidicolin (100 nm) Control No Treatment Aphidicolin Conc. Response 96 well plate readout displaying % cells in S-phase and a heat map display. The top half of the plate contains 100nM Aphidicolin and controls; the bottom is a serial dilution of Aphidicolin across the plate. In the heat map, green represents low % cells in S-Phase, yellow and intermediate % cells in S-phase and red is a high % of cells in S-phase. E ffe c t o f A p h id ic o lin o n C e ll C y c le P o p u la tio n p e r c e n ta g e s IC50 A p h id ic o lin (M ) G0/G1 2.11e-007 S-Phase 2.952e-007 G 0 /G 1 S -P h a s e G 2 /M G2/M 7.449e-007 Celigo Assay Cell cycle analysis using BrdU and DAPI Rev A

26 Assay Name: Cell viability analysis using calcein AM, Hoechst and PI Assay ID: Celigo_02_0002 Description: Analysis of cell viability on A549 cells using calcein AM, Propidium Iodide and Hoechst Stains: calcein AM (green total live cells); Propidium Iodide (red dead cells); Hoechst (blue cell nuclei) Imaging channels: Green, Red, Blue Image analysis algorithm: Celigo software Cell Viability (Live+ Dead + Total) 8. Seed cells in 96-well plate and incubate cells overnight 9. Add drug compound at 2x desired final concentration and incubate for desired time 10. Wash wells with media twice 11. Prepare and add calcein AM, Propidium Iodide and Hoechst 2X mixed dye solution 12. Incubate cells for 30 min 13. Acquire a whole-well image of an entire 96-well plate on Celigo ~ 15 minutes Viability measurement of A549 cells labeled with calcein AM, Propidium Iodide and Hoechst Calcein AM detects enzymatic activity in live cells, Propidium Iodide detects dead cells with compromised membranes and Hoechst is a marker for all nucleated cells. Identification and segmentation of live, dead and all cells are used to report the counts and percentages of live, dead and total numbers of cells. Celigo Assay Cell viability using calcein AM, Hoechst and PI Rev A

27 Control No Treatment Hydrogen Peroxide (12 mm) Hydrogen Peroxide Conc. Response Whole plate readout with the heat map applied. Green: well containing a high % of live cells; Yellow: for wells with an intermediate level of live cells; Red: for a well containing a low % of live cells The plot above was generated by exporting the data into graph pad prism and graphing the percent of viability over the hydrogen peroxide concentration Celigo Assay Cell viability using calcein AM, Hoechst and PI Rev A

28 Assay Name: Endpoint apoptosis assay using Caspase 3/7 with Hoechst Assay ID: Celigo_02_0003 Description: Detect and quantify the number of Caspase 3/7 positive cells in an apoptosis assay Stains: Caspase 3/7 (Green) for detection of apoptotic cells; Hoechst (Blue) for detection of cell nuclei Imaging channels: Bright field, Green, Blue Image analysis algorithm: Celigo software Target Mask 1. Seed MDA-MB-231 cells in a 96-well plate 2. Treat with selected drugs or vehicle control 3. Stain the cells with Caspase 3/7 and Hoechst and incubate for 30 minutes at 37 C (Nexcelom, Cat# CSK- V0003-1) 4. Image entire 96-well plate (whole well imaging) in ~15 minutes 5. Analyze apoptosis data using Celigo gating interface ~ 20 minutes Apoptosis assay using MDA-MB-231 cells with Staurosporine-treated positive control MDA-MB-231 Adherent: Control Scatter Identified Caspase + Caspase Pos cells Results Control Caspase Neg Treated Scatter Plot Identified Caspase + Results 3 µm Staurosporine Caspase Pos Caspase Neg Celigo Assay Endpoint apoptosis assay using Caspase 3/7 with Hoechst Rev B

29 Assay Name: Cell cycle analysis using EdU and DAPI Assay ID: Celigo_02_0004 Description: Measure cell cycle in drug treated and control samples by staining with EdU and DAPI Stains: EdU (green, detects cells in the S-phase), DAPI (blue, detects cells in G 0 /G 1 and G 2 /M phases) Imaging channels: Bright field, Green, Blue Image analysis algorithm: Celigo software Target Seed A549 cells in a 96-well plate 7. Treat with selected drugs or vehicle control 8. Add media with EdU per well 9. Fix the cells in ice cold, 100% Methanol for 15 minutes 10. Stain with DAPI (Nexcelom, Cat# CS1-0127) 11. Image entire 96-well plate (whole well imaging) in ~15 minutes 12. Analyze cell cycle data using Celigo gating interface ~ 20 minutes Cell cycle assay using A375 cells with Staurosporine-treated positive control Control Scatter Plot Identified EdU and DAPI stained cells at different cell cycle stages Results Treated Scatter Plot Identified EdU and DAPI stained cells at different cell cycle stages Results Celigo Assay Cell cycle analysis using EdU and DAPI Rev B

30 Assay Name: Endpoint viability using DRAQ7 and Hoechst Assay ID: Celigo_02_0005 Description: Detect and quantify the number and percentage of DRAQ7-positive cells using Hoechst Stains: DRAQ7 (Far Red, for detection of dead cells); Hoechst (Blue, for detection of total nucleated cells) Imaging channels: Far Red, Bright field, Blue Image analysis algorithm: Celigo software Target Mask 13. Seed MDA-MB-231 adherent cells in a 384-well plate 14. Treat with selected drugs or vehicle control 15. Stain the cells with DRAQ7 and Hoechst after 24 hours of incubation 16. Image entire 384-well plate (whole well imaging) in ~15 minutes 17. Analyze viability images using Celigo gating interface Viability assay using MDA-MB-231 adherent cells treated with Benzethonium and control 25 µm Benzethonium Control Celigo Assay Endpoint viability using DRAQ7 and Hoechst Rev B

31 IC 50 dose-response curves and values for all 3 tested time points Celigo Assay Endpoint viability using DRAQ7 and Hoechst Rev B

32 Assay Name: Kinetic viability using DRAQ7 Assay ID: Celigo_02_0006 Description: Detect and quantify the number of DRAQ7-positive cells in a kinetic viability assay Stains: DRAQ7 (Far Red) for detection of dead cells Imaging channels: Far Red, Bright field Image analysis algorithm: Celigo software Target Seed MDA-MB-231 adherent cells in a 384-well plate 19. Treat with selected drugs or vehicle control 20. Stain the cells with DRAQ7 and incubate for 24, 48 and 72 hours at 37 C, 5% CO Image entire 384-well plate (whole-well imaging) after each time point of 24, 48 and 72 hours 22. Analyze viability images using Celigo to obtain the dead cell counts, ~ 20 minutes Kinetic viability assay using MDA-MB-231 cells treated with Benzethonium drug MDA-MB-231 Adherent: Identified DRAQ7 + cells IC 50 dose response curves and values for all 3 tested time points Image Graphic Overlay Celigo Assay Kinetic viability using DRAQ Rev B

33 Assay Name: HTS suspension cell count and viability using AO/PI Assay ID: Celigo_02_0007 Description: Measure cell count and viability using Acridine Orange (AO) and Propidium Iodide (PI) on suspension cells to increase the efficiency of cell-based analysis for high-throughput screening Stains: Acridine Orange (AO) and Propidium Iodide (PI) Imaging channels: Green and Red Image analysis algorithm: Celigo software Target Culture and collect Jurkat cells 52. Prepare the AO/PI working concentration by diluting AO/PI (Nexcelom, Cat# CS2-0106) by 10X in PBS 53. Pipette 25 µl of AO/PI and 5 µl of Jurkat cells into each well on a 96-well half area microplate Plate 1: Serial dilution of cells. Plate 2: Five viability percentages Dilution Viability A Neat 2X dilutions --> A 100% 75% 50% 25% 0% B Neat 2X dilutions --> B 100% 75% 50% 25% 0% C Neat 2X dilutions --> C 100% 75% 50% 25% 0% D Neat 2X dilutions --> D 100% 75% 50% 25% 0% E Neat 2X dilutions --> E 100% 75% 50% 25% 0% F Neat 2X dilutions --> F 100% 75% 50% 25% 0% G Neat 2X dilutions --> G 100% 75% 50% 25% 0% H Neat 2X dilutions --> H 100% 75% 50% 25% 0% 54. Use Celigo and capture images and analyze total AO and PI positive Jurkat cells in the wells 55. Use the equation below to calculate viability % a. Viability % = 4] ^6_282Y1 01//_ 4]W`a ^6_282Y1 01//_ x 100 Counting AO and PI positive cells using Celigo image cytometer Celigo Assay HTS count and viability using AO/PI Rev A

34 Example AO/PI fluorescent images, and counted images at different Jurkat cell concentrations The fluorescent images showed decreasing number of AO positive cells as the dilution factor increased The correlation plot is the actual counting accuracy against theoretical counts The R 2 value was highly linear (0.9937) Depending on the cell volume pipetted into the well, the concentration range can go from 1 cell to approximately 1 x 10 7 cells/ml The AO/PI fluorescent images showed increase in PI positive cells as the viability decreased The linearity plot is the measurement of cell viability The Celigo can measure accurate cell viability from 0 to 100% The R 2 value was highly linear (0.9977) The Celigo image cytometer was able to perform high-throughput cell count and viability measurement o The method was compared directly to Cellometer image cytometer and was highly comparable Celigo Assay HTS count and viability using AO/PI Rev A

35 Assay Name: p53 and phospho-p53 fluorescent marker analysis Assay ID: Celigo_02_0009 Description: Quantification of p53 and phospho-p53 levels in NCI-H460 cells post Hydroxyurea treatment Stains: CellTracker Blue CMAC, secondary Alexa Fluor 488 antibody, secondary Alexa Fluor 568 antibody Imaging channels: Green, Red and Blue Image analysis algorithm: Celigo software Expression Analysis: Target Mask 56. Stain cells in the tissue culture flask with CellTracker Blue (10 μm final), incubate for 30 min at RT 57. Seed 2,000 cells/well in 384-well plate 58. Add desired drug (Hydroxyurea) or control solution in each well and incubate cells for 24h (or 48h) at 5% CO 2, 37 C 59. Fix and permeabilize the cells 60. Add p53 antibody and phospho-p53 primary antibodies (1:200) and incubate at 4 C overnight 61. Wash plate multiple times and add 10 μl of secondary antibody (1:200) and Incubate for 1 hour at RT 62. Image the plate on the Celigo Detection of phospho-p53 and p-53 detection in NCI-H460 cells post Hydroxyurea treatment Hydroxyurea is an inducer of p53 upregulation and phosphorylation As hydroxyurea concentration increases, we observe an increase in p53 and phospho-p53 signal, and this is depicted in both the image and graphical data above. Celigo Assay p53 and phospho-p53 fluorescent marker analysis Rev A

36 Assay Name: Endpoint viability using PI and Hoechst Assay ID: Celigo_02_0010 Description: Detect and quantify the number and percentage of PI-positive cells using Hoechst Stains: PI (Red, for detection of dead cells); Hoechst (Blue, for detection of total nucleated cells) Imaging channels: Red, Bright field, Blue Image analysis algorithm: Celigo software Target Mask 23. Seed MDA-MB-231 adherent cells in a 384-well plate 24. Treat with selected drugs or vehicle control 25. Stain the cells with PI and Hoechst after 24 hours of incubation 26. Image entire 384-well plate (whole well imaging) in ~15 minutes 27. Analyze viability images using Celigo gating interface Viability assay using MDA-MB-231 adherent cells treated with Benzethonium and control Control Dead PI + 25 µm Benzethonium Dead PI + Celigo Assay Endpoint viability using PI and Hoechst Rev B

37 IC 50 dose response curves and values for all 3 tested time point Celigo Assay Endpoint viability using PI and Hoechst Rev B

38 Assay Name: Kinetic viability using Propidium Iodide Assay ID: Celigo_02_0011 Description: Detect and quantify the number of PI-positive cells in a kinetic viability assay Stains: PI (Red, for detection of dead cells) Imaging channels: Red, Bright field Image analysis algorithm: Celigo software Target Seed MDA-MB-231 adherent cells in a 384-well plate 29. Treat with selected drugs or vehicle control 30. Stain the cells with PI and incubate for 24, 48 and 72 hours at 37 C, 5% CO Image entire 384-well plate (whole well imaging) after each time point of 24, 48 and 72 hours 32. Analyze viability images using Celigo Target 1+2 to obtain the dead cell counts, ~ 20 minutes Kinetic viability assay using MDA-MB-231 cells treated with Benzethonium drug MDA-MB-231 Adherent: BF + Red Image Red Image Images + Graphic Overlay BF Image Celigo Assay Kinetic viability using PI Rev B

39 Assay Name: Kinetic apoptosis using Caspase 3/7 Assay ID: Celigo_02_0012 Description: Kinetic detection of apoptosis in adherent and suspension cells using Nexcelom ViaStain TM Live Caspase 3/7 and bright field imaging. Stains: Nexcelom ViaStain TM Live Caspase 3/7 detection for 2D/3D Culture Imaging channels: Green and Bright field Image analysis algorithm: Celigo software Expression Analysis: Target Adherent cells (MDA-MB-231) 63. Seed 10,000 cells/well in 96-well plate and incubate cells overnight 64. Remove media and add Caspase 3/7 as well as Staurosporine or vehicle control 65. Image at time point 0 and then incubate 66. Image at 2h, 6h and 8h on the Celigo image cytometer Suspension cells (Jurkat) 1. Prepare cell suspension solution of 200 cells/µl in media and add Caspase 3/7 2. Seed 20,000 cells/well in 96-well plate 3. Add 2x Staurosporine (apoptotic inducing drug) or the vehicle control 4. Image at time point 0 and then incubate 5. Image at 2h, 6h and 8h on the Celigo image cytometer Detection of apoptosis in MDA-MB-231 and Jurkat cell using Caspase 3/7 Typical images of Caspase 3/7 labelled apoptotic MDA-MB-231 cells treated with Staurosporine, with or without segmentation, to allow cell identification. Celigo Assay Kinetic apoptosis using Caspase 3/ Rev A

40 MDA-MB-231 Jurkat Whole-plate readout with the heat map applied. Green for wells containing a low number of apoptotic cells, yellow for wells with an intermediate level of apoptotic cells and red for a well containing a high number of apoptotic cells The Nexcelom ViaStain Live Caspase 3/7 detection for 2D/3D Culture can kinetically track the induction of apoptosis by Staurosporine in both MDA- MB-231, an adherent cell line, and Jurkat, a suspension cell line. Celigo Assay Kinetic apoptosis using Caspase 3/ Rev A

41 Assay Name: HPC proliferation measurement using Ki-67 cellular marker Assay ID: Celigo_02_0014 Description: To demonstrate the capability of the Celigo to perform rapid, high throughput imaging and analysis of hematopoietic progenitor cell (HPC) proliferation using the Ki-67 cellular marker. The Ki-67 protein is a biomarker for cell proliferation which are present in all the active phases of cell cycle such as G1, S, G2, and mitosis, but not in G0 phase. Stains: Bright Field, DyLight488 (Green) and DAPI (Blue) Imaging channels: Bright Field, Green, and Blue Image analysis algorithm: Celigo software Target Mask 67. Collect different samples of ipscs isolated from donors 68. Plate HPCs into 6-well plates and incubate for 2 days 69. At end of incubation, fix cells with 4% formaldehyde, permeabilize with 0.2% Triton X Stain cells with primary human anti-ki-67 overnight, then stain with secondary DyLight488 antibody for 1 hour 71. Finally, counterstain the cells with DAPI 72. Image and analyze the stained cells on Celigo for end point reading Celigo-captured bright field, DyLight488 and DAPI fluorescent images Examples of bright field, Ki-67-DyLight488 and DAPI stained fluorescent images Bright Field Ki-67-DyLight488 DAPI Celigo used the counted DAPI-positive cells as the total, then used the gating function to identify Ki-67positive cells and calculate the percent of Ki-67-positive cells for the whole plate Celigo Assay HPC proliferation measurement using Ki-67 cellular marker Rev A

42 Celigo was able to calculate the percent of Ki-67-positive cells for the individual wells of the entire plate Celigo was able to image and identify Ki-67-positive cell population percentages with the Celigo gating function The percent of Ki-67-positive cell population is automatically generated by Celigo software Celigo Assay HPC proliferation measurement using Ki-67 cellular marker Rev A

43 Assay Name: Antibody Cell Surface Binding Detection Assay ID: Celigo_02_0017 Description: Measure R-PE intensity of cell lines that produce CD3 surface markers Stains: Hoechst, Secondary Ab tagged with R-PE (Phyco Erythrin) Imaging channels: Bright field, Red and Blue Image analysis algorithm: Celigo software Target Mask 73. Culture and collect Jurkat and A549 Target cells 74. Seed the Target cells in the wells of 96-well V-bottom microplate containing primary antibody 75. Add secondary antibody tagged with R-PE to the plate 76. Incubate the cells with secondary antibody and Hoechst for an hour. Wash excess stain with PBS Transfer the contents to a flat bottom plate to capture images on Celigo. Analyze the total number of cells expressing CD3 cell surface markers The Hoechst dye is used to identify cell nuclei and an outline is drawn by the Celigo software where the Red R-PE and blue Hoechst fluorescence are measured AVG Red integrated intensity from the exported file: Celigo Assay Antibody Cell Surface Binding Detection Rev A

44 Graph generated by PRISM Graphpad by plotting R-PE Integrated Intensity against Ab concentration: C D 3 c e ll s u rfa c e B in d in g R -P E In te g r a te d In te n s ity J u rk a t C D 3 J u rk a t M O P C 2 1 A C D 3 A M O P C C D 3 (µ g /m l) Celigo Assay Antibody Cell Surface Binding Detection Rev A

45 Assay Name: Kinetic phrodo Antibody Internalization using Confluence Assay ID: Celigo_02_0020 Description: Antibody internalization assays using a ph sensitive dye. Stains: phrodo (ph sensitive dye) Imaging channels: Bright field and Red Fluorescent channel Image analysis algorithm: Celigo software Confluence ratio 78. SKBR-3 and MDA-MB-468 cells were seeded at 10,000 cells/well and left overnight to adhere 79. phrodo-labeled antibodies X, Y and Z were added at 300ng/ml 80. Bright field and fluorescent images were captured at 0, 4, 8 and 24 hrs 81. Kinetic internalization can be tracked using Confluence Ratio application on the Celigo Combined bright field and fluorescent images and numerical data detecting antibody internalization The qualitative phrodo red fluorescent images overlaid with bright field label free imaging show cellular localization of antibodies using the ph-sensitive dye in real time. The quantitative data in the line graphs confirm the previous known activities of antibodies X, Y and Z. Celigo Assay Kinetic phrodo Antibody Internalization using Confluence Rev A

46 Assay Name: Kinetic antibody internalization using phrodo and bright field Assay ID: Celigo_02_0021 Description: Antibody internalisation assays using a ph-sensitive dye. Stains: phrodo (ph sensitive dye) Imaging channels: Bright field and Red Fluorescent channel Image analysis algorithm: Celigo software Target SKBR-3 and MDA-MB-468 cells were seeded at 10,000 cells/well and left overnight to adhere 83. phrodo-labeled antibodies X, Y and Z were added at 300ng/ml 84. Bright field and fluorescent images were captured at 0, 4, 8 and 24 hrs 85. Kinetic internalization can be tracked using Target application on the Celigo Combined bright field and fluorescent images and numerical data detecting antibody internalization The qualitative phrodo red fluorescent images overlaid with bright field label free imaging show cellular localization of antibodies using the ph-sensitive dye in real time. The quantitative data in the line graphs confirm the previous known activities of antibodies X, Y and Z. Celigo Assay Kinetic Antibody Internalization using phrodo and Bright Field Rev A

47 Assay Name: Endpoint antibody internalization using phrodo and Hoechst Assay ID: Celigo_02_0022 Description: Antibody internalisation assays using a ph sensitive dye. Stains: Hoechst and phrodo (ph sensitive dye) Imaging channels: Blue and Red Fluorescent channels Image analysis algorithm: Celigo software Target 1 + Mask 86. SKBR-3 and MDA-MB-468 cells were seeded at 10,000 cells/well and left overnight to adhere 87. phrodo-labeled antibodies X, Y and Z were added at 300ng/ml and incubated for 24 hours 88. Cells were labeled with 1 µg/ml of Hoechst for 30 minutes and read on the Celigo using Target 1 + Mask Combined fluorescent images and numerical data detecting antibody internalisation The qualitative phrodo red fluorescent images overlays with nuclear Hoechst imaging show cellular localization of antibodies using the ph-sensitive dye. The quantitative data in the bar graph confirms the previous known activities of antibodies X, Y and Z. Celigo Assay Endpoint Antibody Internalization using phrodo and Hoechst Rev A

48 Assay Name: Label free tumor spheroid growth inhibition Assay ID: Celigo_03_0001 Description: Measure the size of tumor spheroids treated with drugs and grown under various conditions in a 96-well plate, label free Stains: Label Free Imaging channels: Bright field Image analysis algorithm: Celigo software Tumorsphere Seed 2,500 NCI-H460 cells/well in a ULA 96-well plates 34. On day 4, treat formed spheroids with drug X or vehicle control 35. Place one plate in hypoxic conditions and one plate at standard growth conditions 36. On day 4 post-treatment, image each entire 96-well plate in ~5 minutes Measuring tumor spheroid growth inhibition NCI-H460 Spheroid at Normoxia, treated with Drug X at 10 µm NCI-H460 Spheroid at Hypoxia, treated with Drug X at 10 µm The spheroids were imaged and analyzed using the Celigo image cytometer. The spheroid diameters are automatically reported for each spheroid. Drug-treated spheroids at hypoxic conditions had a smaller spheroid diameter compared to the spheroids grown at normal conditions. Celigo Assay Label free tumor spheroid growth inhibition Rev B

49 Assay Name: 3D multicellular tumor spheroid (MCTS) invasion screening Assay ID: Celigo_03_0002 Description: Monitor the effect of a panel of drugs on the invasion of U87MG Glioblastoma MCTS into Basement Membrane Extract (BME) Matrigel Stains: Label-Free Imaging channels: Bright Field Image analysis algorithm: Celigo software Tumorsphere Migration 37. Seed 500 U87MG cells/well in ULA 384-well plates 38. On day 4, add different drug compounds at 2x or vehicle control in Basement Membrane Extract (BME) Matrigel 39. Monitor invasion by imaging and analyzing each 384-well plate at ~5 minutes/plate at 24, 48 and 72 hours on the Celigo imaging cytometer 40. Measure the invasion area on 24, 48, and 72 hours for each drug compound-treated MCTS 41. Compare the invasion area for each drug compound at each time point to characterize the tested compounds Celigo captured Bright Field images showing invasion of the U87MG MCTS The Celigo produces quantitative data and qualitative images, as displayed above The invasion area observed in the raw images can be outlined using a green fill option, allowing for simple visualization Celigo Assay 3D MCTS invasion screening assay Rev B

50 Kinetic measurement of 3D MCTS invasion inhibition Invasion Area (µm2) Time-Course Monitoring of Invasion Area (5 µm) Time (Hour) Control The plot above was generated by exporting the data from Celigo into Excel and graphing the invasion area over time Kinetic monitoring allows the detection of the invasive inhibitory properties of compounds 10 and 12, which would not be detected in a 24 or 48 hour endpoint assay. Endpoint measurement of 3D MCTS invasion inhibition Average Caspase 3/7 Fluorescent Intensity at 5 µm of Drug Invasion Area (µm 2 ) CNTL Compounds The plot above was an endpoint plot at Day 13. It shows the Control, and Compounds 1, 6, 9, 10, and 12 allowed normal invasion of the spheroids The rest of the drug compounds tested inhibited the invasion of MCTS into BME Matrigel Celigo Assay 3D MCTS invasion screening assay Rev B

51 Assay Name: 3D multicellular tumor spheroid (MCTS) growth inhibition screening assay Assay ID: Celigo_03_0004 Description: Monitor the effect of a panel of drugs on the growth inhibition of U87MG Glioblastoma MCTS Stains: Label-Free Imaging channels: Bright Field Image analysis algorithm: Celigo software Tumorsphere Seed 500 U87MG cells/well in ULA 384-well plates 43. On day 4, add serially diluted different drug compounds at 2x and a vehicle control in media 44. Monitor growth inhibition by imaging and analyzing each 384-well plate at ~4 min/plate on Day 4, 7, 9, 11, and 13 with the Celigo imaging cytometer 45. Measure the spheroid diameters on Day 4, 7, 9, 11, and 13 for each drug compound treated MCTS 46. Compare the spheroid diameters for each drug compound at each time point to characterize the tested compounds Celigo-captured Bright Field images showing growth inhibition of the U87MG MCTS The bright field images were analyzed to measure the spheroid size in diameter for all the MCTS The images above showed that some drug compounds can induce inhibition of spheroid growth Celigo Assay 3D MCTS growth inhibition screening Rev A

52 Kinetic measurement of 3D MCTS growth inhibition Time-Course Monitoring of Spheroid Diameter (5 µm) Spheroid Diameter (µm) Time (Hour) Control The plot above was generated by exporting the data from Celigo into excel and graphing the spheroid diameters over time The plot showed some of the drug compounds did not inhibit spheroid growth while other compounds showed moderate to high growth inhibition Endpoint measurement of 3D MCTS growth inhibition Average Spheroid Diameter at 5 µm of Drug Spheroid Diameter (µm) CNTL Compounds The plot above was an endpoint plot at Day 13 showing Control, 1, 6, 9, and 10 allowed normal growth of the spheroids The rest of the drug compounds tested inhibited the growth of MCTS Celigo Assay 3D MCTS growth inhibition screening Rev A

53 Assay Name: 3D multicellular tumor spheroid endpoint apoptosis screening Assay ID: Celigo_03_0005 Description: Measure the effects of a panel of drugs on the apoptosis of U87MG Glioblastoma MCTS using Caspase 3/7 and Hoechst fluorescent staining Stains: Caspase 3/7 and Hoechst Imaging channels: Green, Blue, and Bright Field Image analysis algorithm: Celigo software Tumorsphere Mask 47. Seed 500 U87MG cells/well in ULA 384-well plates 48. On day 4, add different serially diluted drug compounds at 2x and a vehicle control in media 49. On day 13, prepare and stain the MCTS with the Caspase 3/7 and Hoechst 50. Incubate the plate at 37 C and 5% CO2 for 60 min 51. Image and analyze on Celigo 52. Compare the spheroid apoptosis for each drug compound at each time point to characterize the tested compounds Celigo-captured bright field and fluorescent images showing Caspase 3/7 and Hoechst staining of the U87MG MCTS The bright field images were used to identify the spheroids in the well The caspase 3/7 fluorescent intensities were measured from the images Celigo Assay 3D MCTS endpoint apoptosis screening Rev A

54 Endpoint measurement of 3D MCTS apoptosis Average Caspase 3/7 Fluorescent Intensity at 5 µm of Drug AVE Caspase 3/7 INT (R.U.) CNTL Compounds The plot above shows the Caspase 3/7 fluorescent intensities, which indicate the apoptosis of each MCTS treated with the different drug compounds Only 2 drug compounds induced noticeable apoptosis on the U87MG MCTS, while other drugs had no effect Celigo Assay 3D MCTS endpoint apoptosis screening Rev A

55 Assay Name: 3D multicellular tumor spheroid (MCTS) endpoint viability screening assay Assay ID: Celigo_03_0007 Description: Measure the effects of a panel of drugs on the viability of U87MG Glioblastoma MCTS using calcein AM and Propidium Iodide fluorescent staining Stains: Calcein AM and Propidium Iodide Imaging channels: Green, Red, and Bright Field Image analysis algorithm: Celigo software Tumorsphere Mask 53. Seed 500 U87MG cells/well in ULA 384-well plates 54. On day 4, add different diluted drug compounds at 2x and a vehicle control in media 55. On day 13, prepare and stain the MCTS with the calcein AM and PI 56. Incubate the plate at 37 C and 5% CO2 for 60 min 57. Image and analyze on Celigo 58. Compare the spheroid viability for the tested drug compounds Celigo-captured bright field and fluorescent images showing calcein AM/PI staining of the U87MG MCTS The bright field images were used to identify the spheroids in the well The calcein AM and PI fluorescent intensities were measured from the images Calcein AM/PI intensity ratios were calculated to determine the viability Celigo Assay 3D MCTS endpoint viability screening Rev A

56 Endpoint measurement of 3D MCTS viability Average Calcein AM/PI Ratio at 5 µm of Drug AVE CAM INT/AVE PI INT CNTL Compounds (µm) The plot above is showing the calcein AM/PI fluorescent intensity ratios, which indicates the viability of each MCTS treated with the different drug compounds Few drug compounds had moderate to high cytotoxic effects on the U87MG MCTS, while other drugs had no effects This method allows researchers to quickly identify the effects of the drug compounds on tumor spheroid viability in a high throughput manner Celigo Assay 3D MCTS endpoint viability screening Rev A

57 Assay Name: 3D MCTS Kinetic Propidium Iodide Assay Assay ID: Celigo_03_0008 Description: Monitor the growth and signal intensity of dead cell stain, Propidium Iodide (PI), kinetically on U87MG Glioblastoma multicellular tumor spheroids (MCTS) for 10 days Stains: Propidium Iodide Imaging channels: Bright field, Red Image analysis algorithm: Celigo software Tumorsphere 1 + Mask 59. Seed different cell numbers from 3,200 to 100 U87MG cells/well in ULA 96-well plate 60. Add PI and vehicle control in media to the plate. Spin the plate at 2,000 rpm for 10 min 61. Incubate the plate at 37 C and 5% CO 2 until MCTS are formed 62. Image and analyze on Celigo for 10 days 63. Measure and graph the intensity of PI and diameter of MCTS for 10 days Figure 1. Bright field and red fluorescent images of U87MG MCTS with PI (red) over 10 days Celigo Assay 3D MCTS Kinetic PI Assay Rev A

58 Figure 2. PI dead cell signal intensities and sphere diameter over 10 days Line graph showing the trends of PI intensity for different cell concentration for 10 days (a). Line graph showing that there is no toxic effect of PI on MCTS diameter (b). (a) (b) Conclusion: Using the Celigo image cytometer, images of U87MG MCTS were successfully captured and analyzed for growth and PI dead cell signal intensities Adding PI at the beginning of the experiment allowed for kinetic monitoring of cell death in the core of the MCTS As the sphere diameter begins to plateau, the signal of PI increases The entire 96-well plate was imaged in 2 minutes. The short scan time significantly increases the throughput during an experiment that has multiple plates Celigo Assay 3D MCTS Kinetic PI Assay Rev A

59 Assay Name: Scratch wound healing assay using direct cell counting Assay ID: Celigo_04_0002 Description: Cell counting to measure would healing scratch assay for oncology research Stains: Label Free Imaging channels: Bright field Image analysis algorithm: Celigo software Target Seed PC3 cells and leave overnight to reach confluence 90. Create uniform scratch using P-1000 pipette tip 91. Wash off any floating cells from the scratch wound 92. Capture images at 0, 24 and 48 hrs and count cells in a predefined as displayed below Figure 1. Wound creating diagram Celigo gating interface demonstrating the closure of scratch wound over time Red outline depicts cells within the gate/wound and blue are cells outside the gate/wound Closure of the wound is displayed visually within the gates and numerically in the line graph Celigo Assay Scratch Wound Healing Assay using Direct Cell Counting Rev A

60 Assay Name: Scratch wound healing assay using confluence Assay ID: Celigo_04_0003 Description: Cell counting using confluence to measure would healing scratch assay for oncology research Stains: Label free Imaging channels: Bright field Image analysis algorithm: Celigo software Confluence 93. Seed PC3 cells and leave overnight to reach confluence 94. Create uniform scratch using P-1000 pipette tip 95. Wash off any floating cells from the scratch wound 96. Capture images at 0, 24 and 48 hrs and count cells in a predefined as displayed below. Figure 1. Wound creating diagram Whole-well confluence using a pseudo-green fill overlay demonstrates the closure of scratch wound over time Whole-well readouts using a pseudo-green fill overlay to detect cell confluence Closure of the wound is displayed both visually, at the whole-well level, and numerically, in the line graph Celigo Assay Scratch Wound Healing Assay using Confluence Rev A

61 Assay Name: 2D Cell Migration Assay using 96-well Oris TM Platypus Plate Assay ID: Celigo_04_0004 Description: Cell migration assay is used to study adherent cell migration in a predefined area. Stains: Label-Free Imaging channels: Bright field Image analysis algorithm: Wound Healing 97. Seed adherent HT1080 cells in Oris TM Platypus plate and let adhere overnight. 98. Remove plugs and add drug to wells. 99. Image plate on Celigo at 32 hours after adding drug treatment Analyze captured images for percent wound healing. Images and data from HT1080 cells treated with Cytochalasin D for 32 hours. Bright Field Image Wound Healed Area Cell Counts Control Cytochalasin D (5 µm) W o u n d H e a lin g R o b u s tn e s s H T tr e a te d w ith C y to c h a la s in D 6 0 P e r c e n t W o u n d H e a le d P e r c e n t W o u n d H e a le d Z ' = L o g [u M ] 0 C o n tr o l T r e a te d Celigo Assay 2D Cell Migration Assay with Oris Platypus Plate Rev A

62 Assay Name: Label-free adherent cell proliferation using confluence Assay ID: Celigo_05_0001 Description: Monitor proliferation of adherent cells with confluence area measurements Stains: Label-Free Imaging channels: Bright Field Image analysis algorithm: Celigo software Confluence 101. Culture and collect adherent cells 102. Seed the adherent cells in a 96-well microplate at 2,000 cells/well 103. Centrifuge the microplate using a swing bucket rotor centrifuge for 5 min to settle down all the cells 104. Add drug treatments after overnight incubation 105. Use Celigo and capture images and analyze confluence percentage of adherent cells in the bright field images at multiple time points Measuring confluence of adherent cells using Celigo image cytometer Example of counted adherent cell confluence area at different time points The contrast of the adherent cell membrane allows the Celigo software to accurately count all the cells Celigo Assay Label-free adherent cell proliferation using confluence Rev A

63 By performing confluence measurement on cell samples in different well with drug treatments, a timedependent and dose response graph can be plotted % Confluence Growth curves Hours [Drug A, concentration] Ctrl % Confluence Dose Response [nm] Drug A Drug B Drug C Drug D In addition, multiple time points can be captured by Celigo to create growth tracking of adherent cells over time The method allows high-throughput label-free confluence measurement of adherent cells The Celigo can rapidly and accurately measure confluence in standard multi-well microplates Both drug dose response curves and growth tracking curves can be generated from the measured data Celigo Assay Label-free adherent cell proliferation using confluence Rev A

64 Assay Name: Label-free suspension cell proliferation by direct cell counting Assay ID: Celigo_05_0002 Description: Monitor proliferation of suspension cells with direct cell counting measurements Stains: Label-Free Imaging channels: Bright Field Image analysis algorithm: Celigo software Direct Cell Counting 106. Culture and collect suspension cells 107. Seed the suspension cells in a 96-well microplate at 4,000 cells/well and then add drug treatments 108. Centrifuge the microplate using a swing bucket rotor centrifuge for 5 min to settle down all the cells 109. Incubate the cells overnight 110. Use Celigo and capture images and analyze total number of suspension cells in the bright field images at multiple time points Counting suspension cells using Celigo image cytometer Example of counted suspension cells at different time points The bright center on the suspension cells allows the Celigo software to accurately count all the cells Celigo Assay Label-free suspension cell proliferation by direct cell counting Rev A

65 By performing direct cell counting on cell samples in different well with drug treatments, a dose response graph can be plotted Dose-dependent Total Cell Count Total Cell Count [Drug] (IU/ml) In addition, multiple time points can be captured by Celigo to create growth tracking of suspension cells over time The method allows high-throughput label-free counting of suspension cells The Celigo can rapidly and accurately count suspension cell number in standard multi-well microplates Both drug dose response curves and growth tracking curves can be generated from the measured data Celigo Assay Label-free suspension cell proliferation by direct cell counting Rev A

66 Assay Name: Single Cell Detection of FACS-sorted cells Assay ID: Celigo_07_0001 Description: Single cell detection following serial dilution or single cell fluorescent-activated cell sorting Stains: CellTracker Green (for metabolically active cells) Imaging channels: Bright field and Green Image analysis algorithm: Target Culture and collect CHO-S cells and stain with CellTracker Green (CTG) 112. Seed single cells in the wells of 96-well microplate via serial dilution or fluorescent-activated cell sorting 113. Assign at least one reference well containing 1,000 2,000 cells for focus registration 114. Allow cells to settle for 10 mins or pulse spin using a centrifuge 30 seconds 1,000 rpm 115. Use Celigo to capture images and analyze the total number of live cells per well 96-well microplate heat map showing the number of CTG-labelled cells per well The heat map feature allows for quick visualization of which wells contain single cells, as depicted in yellow. Red wells have more than 1 cell and Green wells contain no cells. Celigo Assay Single cell detection of FACS-sorted cells Rev A

67 Wells C3 and G10 were selected as reference wells for focus registration Whole-well and zoomed images in bright field and fluorescence Confirmation of a single cell in well D7 Confirmation of two cells in well C7 Celigo s whole-well imaging allows for simple validation of successful single cell plating or the presence of more than one cell Celigo Assay Single cell detection of FACS-sorted cells Rev A