HL-60 and THP-1 cells were labeled using CellTrace Far Red Proliferation Kit (Thermo Fisher). Enriched NK cells were plated with targets, indicated treatments and Caspase-3/7 Green Apoptosis Assay reagent (Sartorius Inc., Göttingen, Germany) for reading every 30 minutes in an IncuCyte S3 machine (Sartorius Inc.). A time-dependent graph of the percentage live HL-60 and THP-1 targets (CellTrace Far Red+, Caspase3/7-) was plotted to represent killing after normalization to targets alone.
Incucyte s3 machine
Manufactured by Sartorius
Sourced in Germany
The Incucyte S3 machine is a live-cell analysis system designed for long-term, kinetic monitoring of cell cultures. It provides real-time, automated image acquisition and analysis, enabling researchers to observe and quantify cellular behavior over time without disturbing the cell environment.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace far red proliferation kit, by Thermo Fisher Scientific (2 mentions)
Caspase 3 7 green apoptosis assay reagent, by Sartorius (2 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (2 mentions)
Rna clean and concentrator kit, by Zymo Research (2 mentions)
Cellbrite red, by Biotium (1 mentions)
Cellbrite green, by Biotium (1 mentions)
5 protocols using incucyte s3 machine
1
NK Cell-Mediated Cancer Cell Killing
2
MNV Plasmid Transcription and Transfection
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MNV plasmids were linearised with NotI and phenol/chloroform extracted before being used for in vitro transcription using the HiScribe™ T7 ARCA mRNA Kit (NEB), following the manufacturer’s instructions. RNA was purified and concentrated using the RNA Clean and Concentrator Kit (Zymo). RNA/DNA transfection was carried out as previously described (62 (link)). GFP fluorescence at 24 and 48 hours post transfection was analysed via the Incucyte S3 machine (Sartorius).
3
Macrophage-T Cell Phagocytosis Assay
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MH-S (murine alveolar macrophage) and RAW24.6 (murine macrophage) cell lines were stained with CellBrite Red (Biotium) for 20 min at 37°C. They were then washed and plated at 1.25×105, 6.25×104, or 3.13×104 cells per well in a 48-well tissue culture treated plate and incubated at 37°C for 1–2 hours. NT, control KO (AAVS1-KO), or CD47-KO T cells were then stained with CellBrite Green (Biotium) for 20 min at 37°C. They were then washed and plated on macrophages at a 1 to 1 effector to target ratio. The co-culture was then imaged on a Sartorius Incucyte S3 machine every 20 min for 4 hours. Phagocytosis was determined by analyzing the merge of green and red. All data were normalized to hour 0. To compare across donors, all data were then normalized to NT T cells (of the respective donor). Area under the curve analysis was then performed on the normalized merge data.
4
NK Cell-Mediated Cancer Cell Killing
5
In Vitro Transcription and Transfection of MNV Plasmids
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MNV plasmids were linearized with NotI and phenol/chloroform extracted before being used for in vitro transcription using the HiScribe T7 ARCA mRNA Kit (NEB), following the manufacturer’s instructions. RNA was purified and concentrated using the RNA Clean and Concentrator Kit (Zymo). RNA/DNA transfection was carried out as previously described (76 (link)). GFP fluorescence at 24 and 48 hours post-transfection was analyzed via the Incucyte S3 machine (Sartorius).
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