Protocol
Day 0
- Remove serum-containing medium from culture and gently rinse twice with D-PBS.
NOTE: Culture should be below 80% confluence. A T-25 at 75-80% confluence generally yields enough cells to perform an entire 96-well Chemotaxis Cell Migration Assay.
- Replace with serum-free F12 + 1x Insulin-Transferrin-Selenium (ITS). Return culture to incubator for 20 hours.
Day 1
- In advance, prepare a 3x stock of Cytochalasin D (e.g. highest cell treatment of 3 µM requires a stock concentration of 9 µM) and prepare 3-fold serial dilutions.
- Remove serum-free chemotaxis medium from culture and gently rinse with D-PBS.
- Harvest cells and perform a cell count (e.g., trypan blue staining + hemacytometer). Centrifuge the cell suspension (1000 RPM, 4 minutes) and resuspend the cell pellet in culture medium at 25,000 cells/mL. (Calculation: 25,000 cells/mL x 0.04 mL = 1000 cells per insert well).
NOTE: When performing a chemotaxis cell migration assay without treatment, the cell seeding volume is increased to 60 μL, thus the cell seeding stock is reduced to 16,666.67 cells/mL.
- Using a manual multi-channel pipette and reverse pipetting technique, seed cells (40 µL per well, 1,000 cells per well) into every well of the insert plate. Learn how to seed cells
![icon-video](/media/cache/15/a9/15a922fd4ec87efcd2e8148c102e673b.png)
- Add 20 µL of the prepared Cytochalasin D at 3x final assay concentration to the appropriate wells and mix by repeatedly pipetting up and down with a 30 µL volume.
- Allow the cells to settle at ambient temperature for 15 minutes then place the ClearView Cell Migration Plate at 37°C, 5% CO2 for 30 minutes in order to pre-incubate the HT-1080 cells in the presence of Cytochalasin D.
- Prepare F12 + ITS + 10% FBS as the chemoattractant, and F12 + ITS for the control wells.
- Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir plate.
- Carefully transfer the insert plate containing cells ± Cytochalasin D treatment into the reservoir plate containing medium ± chemoattractant. Learn how to transfer the insert
![icon-video](/media/cache/15/a9/15a922fd4ec87efcd2e8148c102e673b.png)
- Place the ClearView Cell Migration plate into the IncuCyte® ZOOM instrument and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, wipe away any condensation that remains on the outside of the plate lid or bottom of the reservoir.
- In the IncuCyte® ZOOM software; schedule 24 hour repeat scanning (10x) for every 1-2 hours.
- a. Objective: Ensure 10x objective is installed.
- b. Vessel Type: Select ‘ClearView Cell Migration’
- c. Channel Selection: Select ‘Phase’ + ‘Red’ (800 ms acquisition time).
- d. Scan Mode: Select ‘Chemotaxis (Top/Bot)’ scan type and desired Scan Pattern
- e. Note IncuCyte® estimates a scan time of 20 min per plate (phase only) and 33 min per plate (phase and red); however, the actual scan time can take longer if condensation is not properly removed.
Example of Data Generated with the Detailed Adherent Chemotaxis Cell Migration Protocol
![Chemotaxis adherent protocol representative images Representative images of NucLight Red HT-1080 cells migrating toward 10% FBS](/media/cache/35/47/3547b5bae84c7568d6d1158ff58f17cd.png)
Representative images of NucLight Red HT-1080 cells migrating toward 10% FBS. Bottom-side fluorescence image segmentation mask (yellow) is blended with phase-contrast and fluorescence images acquired for NucLight Red HT-1080 cells migrating toward 10% FBS taken at t=0, 24, and 48 hours. Cells were seeded at 500 cells per well into an IncuCyte® ClearView 96-Well Cell Migration Plate.