The method was adapted from Iwancki et al (51 (link)). Glass bottom 8-chamber slides were coated with 0.1 μg/ml fibronectin. Primary FTECs were directly plated into each well (2×104 cells/well) at passage zero and grown to 100% confluency (72 hrs) before labeling with CellTracker Green (1:1000). In parallel, 5×103 TYK-nu cells were plated in a non-adherent 96-well plate (Corning) to form spheroids for 72 hrs. Spheroids were directly transferred from 96-well plate to the FTEC monolayer and incubated for 12 hrs before fixation and staining for actin (AlexaFluor 647 phalloidin, Thermo Scientific). Images were collected on a Zeiss LSM 510 Meta Confocal microscope and images processed with ImageJ.
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Spheroid-Monolayer Co-Culture Assay
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Corresponding Organization : University of Chicago
Other organizations : Northwestern University
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Variable analysis
independent variables
- Coating of glass bottom 8-chamber slides with 0.1 μg/ml fibronectin
- Plating of primary FTECs at passage zero (2×10^4 cells/well)
- Formation of TYK-nu cell spheroids (5×10^3 cells) in non-adherent 96-well plates for 72 hrs
- FTEC monolayer formation (100% confluency after 72 hrs)
- Labeling of FTECs with CellTracker Green (1:1000)
- Transfer of TYK-nu cell spheroids to FTEC monolayer and incubation for 12 hrs
- Fixation and staining of co-culture for actin (AlexaFluor 647 phalloidin)
- Passage zero of primary FTECs
- Incubation time for FTEC monolayer formation (72 hrs)
- Incubation time for TYK-nu cell spheroid formation (72 hrs)
- Incubation time for co-culture (12 hrs)
- Imaging platform (Zeiss LSM 510 Meta Confocal microscope)
- Image processing software (ImageJ)
- No positive or negative controls were explicitly mentioned in the protocol.
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