Microfluidic migration devices with precisely defined constrictions were prepared as described previously25 (link),46 (link). Devices were coated with 50 μg/mL of type-I rat tail collagen (BD Biosciences) in 0.02N acetic acid overnight at 4°C. Approximately 80,000 cells were seeded (in DMEM supplemented with 10% FBS and 1% PenStrep) per migration chamber. Devices were placed in a tissue-culture incubator (37°C) for 5–6 hours to allow the cells to adhere. Subsequently, media was changed to phenol-red free Leibovitz L15 media supplemented with 10% FBS and 1% PenStrep before mounting the device on an inverted microscope (Zeiss Observer Z1) equipped with temperature-controlled stage (37°C) for live-cell imaging. The media reservoirs of the device were covered with glass coverslips to minimize evaporation during live-cell imaging. Cells were imaged for 14–16 hours at 10 minute intervals with a CCD camera (Photometrics CoolSNAP KINO) using a Zeiss 20′/NA 0.8 air objective. Acquired image sequences were analyzed for nuclear rupture frequency, duration, and transit time of cells through 1×5μm2, 2×5μm2, and 15×5μm2 constrictions using Zen (Zeiss) software and a custom-written MATLAB 2016a script for automated image analysis.
Protocol detail
Microfluidic Devices for Cell Migration
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Acetic acid
Cells
Constrictions
Device
Leibovitz l15
Microfluidic devices
Microscope
Tail
Tissue
Type i collagen
Corresponding Organization :
Other organizations : Memorial Sloan Kettering Cancer Center, Cornell University, Ludwig Cancer Research, University of California, San Diego, The Francis Crick Institute, University Hospital of Zurich, Broad Institute, CRUK Lung Cancer Centre of Excellence, University College London, Moffitt Cancer Center
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Variable analysis
independent variables
- Constriction size (1×5μm^2, 2×5μm^2, and 15×5μm^2)
- Nuclear rupture frequency
- Nuclear rupture duration
- Cell transit time through constrictions
- Coating of migration devices with 50 μg/mL of type-I rat tail collagen in 0.02N acetic acid overnight at 4°C
- Seeding of approximately 80,000 cells per migration chamber in DMEM supplemented with 10% FBS and 1% PenStrep
- Incubation of devices in a tissue-culture incubator (37°C) for 5–6 hours to allow cell adhesion
- Changing media to phenol-red free Leibovitz L15 media supplemented with 10% FBS and 1% PenStrep before live-cell imaging
- Live-cell imaging at 37°C using an inverted microscope (Zeiss Observer Z1) with a temperature-controlled stage
- Covering the media reservoirs with glass coverslips to minimize evaporation during live-cell imaging
- Imaging cells for 14–16 hours at 10-minute intervals using a CCD camera (Photometrics CoolSNAP KINO) and a Zeiss 20′/NA 0.8 air objective
- Positive control: Not mentioned
- Negative control: Not mentioned
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