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Incucyte image analysis software

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The IncuCyte image analysis software is a core function tool designed for high-content live-cell imaging and analysis. It provides real-time, automated quantification of various cellular parameters, enabling researchers to monitor and analyze cellular behavior in a non-invasive manner.

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Lab products found in correlation

Sytox green, by Thermo Fisher Scientific (5 mentions) Incucyte flr imaging system, by Sartorius (5 mentions) Sytox green, by Sartorius (3 mentions) Puromycin, by Mirus Bio (2 mentions) Polybrene, by Santa Cruz Biotechnology (2 mentions) Incucyte nuclight red lentivirus reagent, by Sartorius (2 mentions) Incucyte zoom system, by Sartorius (2 mentions) Ferhonox 1, by Goryo Chemical (2 mentions) Hydrop, by Goryo Chemical (2 mentions) Celltiter glo 2.0 cell viability assay kit, by Promega (1 mentions) Incucyte zoom, by Sartorius (1 mentions) Fluostar omega plate reader, by BMG Labtech (1 mentions) Incucyte flr, by Sartorius (1 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions) Cellquest pro software, by BD (1 mentions) Facscalibur, by BD (1 mentions) Incucyte zoom imaging system, by Sartorius (1 mentions) Incucyte s3 imaging system, by Sartorius (1 mentions) Yoyo 1, by Thermo Fisher Scientific (1 mentions) Nuclightred, by Sartorius (1 mentions)

Close spelling variants of this product

IncuCyte (373 citations) IncuCyte software (87 citations) IncuCyte Analysis Software (13 citations)

13 protocols using incucyte image analysis software

1

Cell Viability and Growth Assay Protocol

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Cell viability was measured using the CellTiter-Glo® 2.0 Cell Viability Assay kit (Promega, PR-G7572) according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well culture plates at a density of ~1000 cells/well with 100μl of growth media and incubated at 37°C. 24 hours after seeding, cells were treated with drugs at 10 different doses or IC50 in growth medium. 72 hours after treatment, 100ul of CellTiterGlo reagent was added to each well and incubated for 10 minutes at room temperature protected from light. Then, luminescence was measured using a FLUOstar Omega plate reader (BMG Labtech). Cell growth was assessed by measuring cell culture well confluence using an IncuCyte ZOOM (ESSEN BIOSCIENCE). Cells were seeded at 1 × 103 per well in a 96-well plate, which was loaded in the IncuCyte ZOOM system. Live-cell images were obtained every 4 hours using a 10x objective lens (four images per well) within the instrument. Cell confluence was analyzed using IncuCyte image analysis software (Essen Bioscience).
Baek M.L., Lee J., Pendleton K.E., Berner M.J., Goff E.B., Tan L., Martinez S.A., Mahmud I., Wang T., Meyer M.D., Lim B., Barrish J.P., Porter W., Lorenzi P.L, & Echeverria G.V. (2023). Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment. Oncogene, 42(14), 1117-1131.
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2

Quantitative Cell Death Assay

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Venus-BOK stabilization and cell-death kinetics were monitored by the IncuCyte FLR or Zoom imaging system (Essen Bioscience). Dead cells were stained with 400 ng/mL propidium iodide (PI) or 25 nM SYTOX Green (Invitrogen). In cases where percentages are indicated, total cell number was quantified using SYTO16 Green. SYTOX Green-, PI-, SYTO16 Green-, or Venus-positive cells were quantified by the IncuCyte image analysis software (Essen Bioscience). Data were expressed as positive events per well or as percentages of the ratio of PI to SYTO16 Green counts (% cell death). IncuCyte data are representative of 3 independent experiments. Error bars represent the standard deviation from the mean for triplicate samples.
Llambi F., Wang Y.M., Victor B., Yang M., Schneider D.M., Gingras S., Parsons M.J., Zheng J.H., Brown S.A., Pelletier S., Moldoveanu T., Chen T, & Green D.R. (2016). BOK Is a Non-Canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation. Cell, 165(2), 421-433.
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3

Evaluating Cell Viability and Protein Stability

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For protein stability assays cells were treated with 1mg/ml cycloheximide (Sigma, C7698-1G), 10 μM MG132 (Calbiochem, 474790) or 10 μM lactaystin (Enzo, BML-PI104-1000) for 8 h unless otherwise indicated. Before starvation with HBSS (Gibco, 14025-050), cells were washed for 4 times with PBS. Actinomycin D (Calbiochem, 114666) was used at a concentration of 1 μM. Cell viability was determined using an Incucyte FLR imaging system (Essen Bioscience, Ann Arbor, MI). Cells were plated in medium containing 30 nM SYTOX Green (Invitrogen, S7020). Cells were treated as described, imaged every 30 min over a period of 3 d, and analyzed using Incucyte image analysis software (Essen Bioscience). For quantification, the SytoxGreen fluorescence was normalized to the confluency factor of the respective well and the percentage of SYTOX Green-positive cells was calculated using the maximum SYTOX Green fluorescence at 100% cell death. Alternatively, cell viability was analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Jose, CA). For this purpose, cells were harvested following 24 h of treatment as indicated and stained with Alexa Fluor 647-ANXA5/annexin V (BioLegend, 640911) and 1 μg/ml propidium iodide according to the manufacturer's protocols. Analysis was performed using Cellquest Pro software (BD Biosciences).
Haller M., Hock A.K., Giampazolias E., Oberst A., Green D.R., Debnath J., Ryan K.M., Vousden K.H, & Tait S.W. (2015). Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity. Autophagy, 10(12), 2269-2278.
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4

Cell Viability and Proliferation Imaging

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Cell viability was determined using an Incucyte Zoom imaging system (Essen Bioscience). Cells were plated in medium containing 30 nM SYTOX Green (Life Technologies, S7020). Cells were then treated with doxycycline (1 μg/ml), actinomycin D (1 μM) or conditioned media from either apoptotic or CICD cells and then imaged every 60 or 120 min. The analysis was done using Incucyte image analysis software (Essen Bioscience). For the proliferation assays, the H2B-mCherry expressing cells were incubated with either apoptotic or CICD media and the number of H2B-mCherry positive nuclei was automatically counted over time. For quantification, the number of SYTOX Green or H2B-mCherry positive cells was normalized to the initial confluency factor of the respective well.
Roumane A., Berthenet K., El Fassi C, & Ichim G. (2018). Caspase-independent cell death does not elicit a proliferative response in melanoma cancer cells. BMC Cell Biology, 19, 11.
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5

Quantifying Cell Death Kinetics

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Cell-death kinetics were monitored by the IncuCyte S3 imaging system (Essen Bioscience). Dead cells were stained with 400 ng/ml propidium iodide (PI) or 25 nM SYTOX Green (Invitrogen). SYTOX Green- or PI-positive cells were quantified by the IncuCyte image analysis software (Essen Bioscience). Data were expressed as positive events per well. Error bars represent the SD from the mean for triplicate samples.
Kalkavan H., Chen M.J., Crawford J.C., Quarato G., Fitzgerald P., Tait S.W., Goding C.R, & Green D.R. (2022). Sublethal Cytochrome-c release generates drug-tolerant persister cells. Cell, 185(18), 3356-3374.e22.
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6

Real-Time Cell Viability Assay

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Cell viability was determined using an IncuCyte Zoom imaging system (Sartorius). Cells were seeded onto Imagelock 96-well plates in media containing 30 nM SYTOX Green (Life Technologies, S7020). Following different apoptotic treatments, the cells were imaged every 60 or 120 minutes and the analysis of the SYTOX Green-positive cells was performed using the available IncuCyte image analysis software (Essen Bioscience).
Coursier D., Coulette D., Leman H., Grenier E, & Ichim G. (2023). Live-cell imaging and mathematical analysis of the "community effect" in apoptosis. Apoptosis : an international journal on programmed cell death, 28(3-4).
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7

Tracking Cell Proliferation with IncuCyte

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All cell lines were transduced with IncuCyte NucLight Red lentivirus reagent (EF-1 Alpha promoter, puromycin selection) (Cat # 4476, Sartorius, Ann Arbor, MI, USA). Briefly, cells were seeded and allowed to adhere for 24 h to reach approximately 20%–40% confluence. NucLight reagent was diluted in DMEM containing 8 μg/mL polybrene (Cat # sc-134220, Santa Cruz Biotechnology, Dallas, TX) and added to cells for 48 h. Media was then replaced with fresh DMEM containing 3 μg/mL puromycin (Cat # MIR 5940, Mirus Bio, Madison, WI). Red fluorescence was monitored on the IncuCyte ZOOM system (Sartorius, Ann Arbor, MI), and analyzed using the IncuCyte image analysis software (Sartorius, Ann Arbor, MI). Similarly, shPHGDH cells were generated using lentiviral transduction particles (shPHGDH_1, TRCN0000028520, Sigma Aldrich, St. Louis, MO, USA; shPHGDH_2, TRCN0000233033, Sigma Aldrich, St. Louis, MO, USA). shGFP vectors were utilized as transduction controls. Knockdown of PHGDH was validated using the Wes Automated Western Blotting System (ProteinSimple, Bio-Techne, Minneapolis, MN), which uses automated capillary technology to measure protein expression and total protein concentration, per manufacturer’s protocol.
Rathore R., Caldwell K.E., Schutt C., Brashears C.B., Prudner B.C., Ehrhardt W.R., Leung C.H., Lin H., Daw N.C., Beird H.C., Giles A., Wang W.L., Lazar A.J., Chrisinger J.S., Livingston J.A, & Van Tine B.A. (2021). Metabolic compensation activates pro-survival mTORC1 signaling upon 3-phosphoglycerate dehydrogenase inhibition in osteosarcoma. Cell reports, 34(4), 108678.
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8

Tracking Cell Proliferation with IncuCyte

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All cell lines were transduced with IncuCyte NucLight Red lentivirus reagent (EF-1 Alpha promoter, puromycin selection) (Cat # 4476, Sartorius, Ann Arbor, MI, USA). Briefly, cells were seeded and allowed to adhere for 24 h to reach approximately 20%–40% confluence. NucLight reagent was diluted in DMEM containing 8 μg/mL polybrene (Cat # sc-134220, Santa Cruz Biotechnology, Dallas, TX) and added to cells for 48 h. Media was then replaced with fresh DMEM containing 3 μg/mL puromycin (Cat # MIR 5940, Mirus Bio, Madison, WI). Red fluorescence was monitored on the IncuCyte ZOOM system (Sartorius, Ann Arbor, MI), and analyzed using the IncuCyte image analysis software (Sartorius, Ann Arbor, MI). Similarly, shPHGDH cells were generated using lentiviral transduction particles (shPHGDH_1, TRCN0000028520, Sigma Aldrich, St. Louis, MO, USA; shPHGDH_2, TRCN0000233033, Sigma Aldrich, St. Louis, MO, USA). shGFP vectors were utilized as transduction controls. Knockdown of PHGDH was validated using the Wes Automated Western Blotting System (ProteinSimple, Bio-Techne, Minneapolis, MN), which uses automated capillary technology to measure protein expression and total protein concentration, per manufacturer’s protocol.
Rathore R., Caldwell K.E., Schutt C., Brashears C.B., Prudner B.C., Ehrhardt W.R., Leung C.H., Lin H., Daw N.C., Beird H.C., Giles A., Wang W.L., Lazar A.J., Chrisinger J.S., Livingston J.A, & Van Tine B.A. (2021). Metabolic compensation activates pro-survival mTORC1 signaling upon 3-phosphoglycerate dehydrogenase inhibition in osteosarcoma. Cell reports, 34(4), 108678.
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9

Nuclei Staining and Surface Expression

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Nuclight Red cell lines were treated as previously described and incubated with FabFluor-488 antibody labeling reagents per manufacturer's instructions (4743, Sartorius). hENT1 (sc-4849, Santa Cruz). Surface expression was quantified using an IncuCyte FLR imaging system (Sartorius), imaged every hour over a period of 3 days, and analyzed using IncuCyte image analysis software (Sartorius). Representative pictures were taken from image analysis software.
Prudner B.C., Rathore R., Robinson A.M., Godec A., Chang S.F., Hawkins W.G., Hirbe A.C, & Van Tine B.A. (2019). Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(16), 5122-5134.
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10

Cell Line Colony Formation Assay

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Nuclight Red cell lines were seeded at 200 cells/6 well (triplicates) in 3 mL DMEM. Cells were treated with combination treatment (DTX:GEM:ADI) for 24 hours after treatment media were replaced with fresh media, and cells were incubated for 2 weeks in a humidified incubator with 95% air/5% CO2 at 37° C. Thereafter, cells were analyzed using IncuCyte image analysis software (Sartorius). For quantification of colonies, Nuclear Red counts were calculated for each respective well.
Prudner B.C., Rathore R., Robinson A.M., Godec A., Chang S.F., Hawkins W.G., Hirbe A.C, & Van Tine B.A. (2019). Arginine Starvation and Docetaxel Induce c-Myc-Driven hENT1 Surface Expression to Overcome Gemcitabine Resistance in ASS1-Negative Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(16), 5122-5134.
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