muvicyte  (Revvity)

Journal: In Vitro Models

Article Title: Novel application of live imaging to determine the functional cell biology of endothelial-to-mesenchymal transition (EndMT) within a liver-on-a-chip platform

doi: 10.1007/s44164-022-00034-9

Figure Lengend Snippet: A Schematic of liver-on-a-chip (LOAC) co-culture using human LSECs and HepG2 human hepatocyte cell line. LSECs were co-transduced with CNN1-Rep and constitutive RFP reporter 24 h prior to seeding on LOAC. Cells were allowed to acclimatise under flow for 24 h prior to treatments being added to media reservoirs for 24–72 h. B Treated LOAC were transferred to MuviCyte (Perkin Elmer) in incubator fluorescent microscope to obtain brightfield and multichannel z-stack images of LSECs and hepatocyte layers. C MuviCyte software enables programmable single-field imaging and stitched whole LOAC imaging. CNN1-Rep reporter allows live imaging and quantification of whole LOAC induction of EndMT following 48 h of 10 ng/ml TGFβ2 and 1 ng/ml TNFα. D Whole mount imaging captured by confocal microscopy (Zeiss LMS 800) of fixed cells allows high-resolution comparison of CNN1-Rep + LSECs with healthy RFP + LSECs while counterstaining with ERG (white) and DAPI (blue) confirm TGFβ2 + TNFα induction of CNN1-Rep. + EndMT (scale 100 µm)

Article Snippet: Images of each cellular layer and reporter were then acquired at baseline (days 2/3 post-seeding) and 48 h post-incubation with TGFβ2 and TNFα (10 ng/ml and 1 ng/ml respectively; days 4/5 post-seeding) to induce EndMT using the MuviCyte Live-Cell Imaging System (PerkinElmer, Waltham, MA, USA).

Techniques: Co-Culture Assay, Transduction, Microscopy, Software, Imaging, Confocal Microscopy, Comparison

Journal: In Vitro Models

Article Title: Novel application of live imaging to determine the functional cell biology of endothelial-to-mesenchymal transition (EndMT) within a liver-on-a-chip platform

doi: 10.1007/s44164-022-00034-9

Figure Lengend Snippet: A Schematic of time-lapse live cell imaging of LOAC. The ‘ Pod imaging adapter’ for the automatic MuviCyte stage enables accurate field acquisition for 2 LOACs at the same time, of multiple regions of interest either as individual areas or stitched images in single depth or using Z-stack (recommended) in Tiff file format. MuviCyte image tools (PerkinElmer) enables repetitive imaging of each field at high frequency (Z-stack of 10 images with 3 channels ~ 5 min; therefore, one image cycle 4 fields for 2 LOACs requires ~ 1 h between each allotted time point). Once all fields and time points are captured, image files were transferred to Volocity software to create a first a multichannel Z-stack and then a time-course sequence to generate time-lapse videos. Volocity software provides modular plugins to identify objects, filter by size, and track by frame providing extensive single-cell tracking data. For migration studies, we have focused on the parameters of total distance travelled (μm), displacement (μm), and velocity (μm/time) for either individual or meaned datasets per field. B Static quantitation of CNN1-Rep + EndMT induction between treatments. C Still from 24-h time-lapse imaging showing of individual cell tracking data for CNN1-Rep + EndMT cells following 48-h treatment with a combination of FAA and TGFβ2 + TNFα to model NASH-like microenvironment. D By performing quantitative cell tracking in both ‘healthy’ constitutive RFP + LSECs and eGFP CNN1-Rep + EndMT LSECs with, we evaluated the migratory behaviour of these different cell populations under the same environmental conditions per field ( n = 8 constituting 4 fields on 2 separate LOAC). ** P < 0.01 or *** P < 0.001 by unpaired T -test between groups

Article Snippet: Images of each cellular layer and reporter were then acquired at baseline (days 2/3 post-seeding) and 48 h post-incubation with TGFβ2 and TNFα (10 ng/ml and 1 ng/ml respectively; days 4/5 post-seeding) to induce EndMT using the MuviCyte Live-Cell Imaging System (PerkinElmer, Waltham, MA, USA).

Techniques: Live Cell Imaging, Imaging, Software, Sequencing, Single Cell Tracking, Migration, Quantitation Assay, Cell Tracking Assay