In a 96 well plate (cat# 167008, Thermo Fisher Scientific), 100 μL of media containing primary human T cells with or without CD27xEGFR (10 µg/mL) were added to 100 μL of media containing 2x103 ES-2scFvCD3, DLD-1scFvCD3, FaDuscFvCD3, OVCAR-3scFvCD3, or OVCAR-3scFvCD3EGFRKO cells incubated overnight in the E:T ratios indicated (0:1, 1:1, 2:1, 5:1, 10:1, 20:1). Experiments were imaged for mCherry fluorescence for up to 7 days using the Incucyte S3 live-imaging system (Essen BioScience, Royston, UK) and analyzed using Incucyte S3 software v2021A. Four pictures of each well for each of three technical replicates were acquired and analyzed based on the Top-Hat segmentation method (Radius 50 µm, Threshold 0.0950, Edge Split On, Edge Sensitivity 5, Hole Fill 0 µm2, Adjusted Size 7 pixels, Filters: min Area 210 µm2, min Integrated Intensity 50). As a measure of cytotoxicity, cell survival was calculated as the mCherry area (µm²/image) from the sample at the indicated time point/mCherry area (µm²/image) from the cancer cells only control at the indicated time point. To evaluate direct EGFR-blocking anti-carcinoma activity of CD27xEGFR, 2x103 mCherry expressing FaDuscFvCD3 cells were seeded in a 96 well plate (cat# 167008, Thermo Fisher Scientific) and treated with CD27xEGFR (10 µg/mL) or mAb425 (10 µg/mL) for 3 days.
Incucyte s3 live imaging system
Manufactured by Sartorius
Sourced in United Kingdom, Germany
The IncuCyte® S3 live imaging system is a cell imaging platform designed for real-time, kinetic monitoring of cell processes and responses in live cell cultures. The system captures high-quality images over extended time periods, enabling researchers to analyze cell behavior and dynamics without disturbing the sample.
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Lab products found in correlation
Isotype control antibody, by BioLegend (1 mentions)
Mouse ifn γ, by Miltenyi Biotec (1 mentions)
Incucyte cytotox green dye, by Sartorius (1 mentions)
Incucyte nuclight rapid red dye, by Sartorius (1 mentions)
Texmacs medium, by Miltenyi Biotec (1 mentions)
Cellbind, by Corning (1 mentions)
Apotox glo, by Promega (1 mentions)
Caspase 3 7 green apoptosis assay reagent, by Sartorius (1 mentions)
Glomax discover system, by Promega (1 mentions)
7 protocols using incucyte s3 live imaging system
1
Evaluating Cytotoxic CD27xEGFR Activity Against Cancer Cells
2
High-throughput screening of neurodevelopmental genes
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Cells were imaged using an IncuCyte S3 live imaging system (Essen BioScience) For each experiment, dCas9-KRAB LUHMES were infected in duplicate or triplicate with individual gRNAs and were plated in duplicate or triplicate in wells of a 24-well plate in either self-renewing or differentiation conditions. Nine fields per well were imaged every 4 h for either 3 or 5 d for proliferation or differentiation, respectively. These experiments were repeated two or three times for each individual gRNA. Images were analyzed using the IncuCyte Software. Specifically, we performed the proliferation analysis and NeuroTrack neurite tracing analyses with default parameters. Cell bodies and neurites were detected from phase contrast images. Representative images are shown in Supplemental Figure S7 .
3
Co-culture Spheroid Formation and Live Imaging
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Co-culture spheroids were generated starting from isolated WJ-MSCs stained using Vybrant™ DiD Cell-Labeling Solutions (Molecular Probes, Eugene, OR, USA) and AECs stained using Vybrant™ DiO Cell-Labeling Solutions (Molecular Probes, Eugene, OR, USA). Biefly, 1 × 106 cells/mL in serum-free DMEM were incubated for 20 min at 37 °C with the corresponding labelling solution (5 μL/mL). Cells were washed twice in PBS and seeded in 96-well round-bottom cell-repellant plates. Immediately after seeding, the plates were centrifuged at 50× g for 3 min and placed in the IncuCyte® S3 live imaging system (Sartorius) for 96 h at 37 °C in 5% CO2. Bright-field and immunofluorescence images were acquired using 10× magnification every 3 h from the seeding to the complete spheroid formation after 96 h.
4
Live-Cell Imaging of Transfected Cells
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Transfected cells were seeded at 2.5 × 103 cells/well in a 96-well plates and imaged in an Incucyte S3 Live-Imaging System (Sartorius). Images were captured every 2 h at 10× magnification (Incucyte 10× lens - NA: 0.3) for 3 days, and analysed using the Incucyte S3 software (version 2018c, 2019b and 2020c were used over the course of the experiments).
5
MHC-II Blockade on Tumor Cytotoxicity
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To achieve an effector-to-target (E:T) ratio of 10:1, 10,000 Tsc2-deficient 105K cells were plated in 96 well plates, pre-treated with 10 ng/ml of mouse IFN-γ (Miltenyi Biotech, Cat#130-105-774), and Incucyte Nuclight Rapid Red Dye (1:500 dilution, Sartorius, Cat#4717) for 48 hr before addition of tumor-infiltrating T cells. Each well contained 100 ul of medium supplemented with 1 µl/well of Incucyte Cytotox Green Dye (Sartorius, Cat#4633). For MHC-II blocking experiments, 10 µg/ml of isotype control antibody (BioLegend, Cat#400601) or MHC-II antibody (BioLegend, Cat#107601) was added to each well pre-plated with Tsc2-deficient 105K cells and cultured for 2 hr before addition of CD45+CD3+CD4+CD25-CD38+CD39+ TILs. Cell culture was monitored by the IncuCyte S3 Live Imaging system (Sartorius) at 1-hr intervals for up to 48 hr when needed. All experiments were carried out with samples from each group pooling from 10 independent tumors. The number of dying tumor cells was determined by co-localization of the Cytotox Green signal to tumor cells. Any out of focus frames were discarded.
6
CAR T-Cell Cytotoxicity Assay with Flt3Lg
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CAR T-cells were transferred to IL-7- and IL-15-deficient TexMACS medium (‘Miltenyi Biotec’, Bergisch Gladbach, Germany) and incubated for 3 d until rested.
A 96-well plate was pre-coated with 10 µg/mL of human plasma fibronectin (‘IMTEK’, Moscow, Russia) for 1 h at room temperature. The wells were washed with DPBS (‘HyClone’, Logan, UT, USA). CAR T-cells were transferred to RPMI complete medium. CAR T-cells were mixed with THP-1-mKate2 cells and U937-mKate cells at an E:T ratio of 1:1 or 5:1 and incubated for 4 d (37 °C, 5% CO2). The number of red objects (mKate2-fluorescent target cells) was analyzed every 4 h using an IncuCyte S3 Live Imaging System (‘Sartorius’, Goettingen, Germany).
CAR Jurkat cells were mixed with THP-1-mKate2 cells at an E:T ratio of 1:1, 10:1, or 20:1, incubated, and analyzed similarly.
Soluble recombinant Flt3Lg (‘SCI-Store’, Moscow, Russia) and Flt3Lg-L27P were added to the co-culture medium before cell suspension mixing to the final concentrations of 0.2 ng/mL, 2 ng/mL, 20 ng/mL, and 100 ng/mL.
A 96-well plate was pre-coated with 10 µg/mL of human plasma fibronectin (‘IMTEK’, Moscow, Russia) for 1 h at room temperature. The wells were washed with DPBS (‘HyClone’, Logan, UT, USA). CAR T-cells were transferred to RPMI complete medium. CAR T-cells were mixed with THP-1-mKate2 cells and U937-mKate cells at an E:T ratio of 1:1 or 5:1 and incubated for 4 d (37 °C, 5% CO2). The number of red objects (mKate2-fluorescent target cells) was analyzed every 4 h using an IncuCyte S3 Live Imaging System (‘Sartorius’, Goettingen, Germany).
CAR Jurkat cells were mixed with THP-1-mKate2 cells at an E:T ratio of 1:1, 10:1, or 20:1, incubated, and analyzed similarly.
Soluble recombinant Flt3Lg (‘SCI-Store’, Moscow, Russia) and Flt3Lg-L27P were added to the co-culture medium before cell suspension mixing to the final concentrations of 0.2 ng/mL, 2 ng/mL, 20 ng/mL, and 100 ng/mL.
7
Apoptosis Assay of NF1 Tumor Cell Lines
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Cells were seeded in a 384-well CellBIND (Corning) at 1,000 cells/well with phenol red-free growth media and incubated overnight (~12-14h) at 37°C, 5% CO 2 . The cells were then treated with drug treatments at the concentrations listed as well as Caspase 3/7 Green Apoptosis Assay Reagent (Sartorius) at a 1:1000 dilution (n=4 wells/treatment). Cells were imaged using the Incucyte S3 live imaging system (Sartorius). Phase and green contrast images were captured every 4h for 72h. Images were analyzed using the integrated software. Primary human VS cells were seeded in 384-well CellBIND plates (Corning) at 1,000 cells/well with Schwann media and incubated overnight at 37°C and 5% CO 2 . Cells were then treated with 0.05% dimethyl sulfoxide (DMSO; control condition), omipalisib (2, 4, 8nM), dasatinib (200, 400, 800nM), or the combination in maintenance media consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Seradigm) and 1% penicillin-streptomycin (n=6 per condition). At 96 hrs, the ApoTox-Glo (Promega) assay was used to detect live cell protease activity for viability and cleaved caspase-3/7 activity for apoptosis using the Glomax Discover System (Promega).
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