The following protocol is a detailed example designed to enable you to run a successful transmembrane cell invasion assay using the IncuCyte® Chemotaxis Cell Invasion Assay. Note that the protocol does not include a description of any experiments required for optimization. Here we specifically describe the use of the IncuCyte® ZOOM instrument for establishing and quantifying chemotactic invasion of HT-1080 cells.
1) Remove serum-containing medium from culture and gently rinse twice with D-PBS.
NOTE: Culture should be below 80% confluence. A T-25 flask at 75–80% confluence generally yields enough cells to perform an entire 96-well
2) ChemotaxisCell Migration or Invasion Assay. Replace with serum-free assay medium, F12 + 1x Insulin-Transferrin-Selenium (ITS). Return culture to incubator for 20 hours to serum starve the cells.
1) Thaw Basement Membrane Extract (BME) on ice or in a pre-chilled CoolBox. Once thawed keep at on ice until ready to use.
Note: BME will gel at temperatures above 8°C. It is extremely important to keep this reagent at 4°C to prevent polymerization during assay set up.
1) Pre-cool a ClearView Chemotaxis plate in a CoolBox system containing a frozen cold pack and CoolSink plate (4°C), for 5 minutes.
Note: It is important to keep close temperature control of the ClearView Chemotaxis plate. The IncuCyte® Cell Invasion Kit (Cat. No. 4444) includes a specialized CoolBox system to ensure the temperature of your assay plate and biomatrix materials are maintained between 4-8°C – preventing premature polymerization and eliminating edge effects. Crushed ice can be used as an alternative however non-uniform cooling can lead to assay variability.
2) Add 150 µL of D-PBS (4°C) to each well of the pre-chilled ClearView reservoir plate. Replace the ClearView insert and allow the membrane to prime for 20 minutes within the CoolBox.
3) While the ClearView plate is priming, dilute the BME to 5 mg/mL using chilled assay medium.
Note: This reagent will be used at a volume of 20 µL per insert well, but prepare the volume you need with excess (e.g., prepare 4 mL biomatrix solution to provide 20 µL per insert well).
4) Remove medium from culture and gently rinse with D-PBS. Harvest cells and perform a cell count (e.g., trypan blue staining + hemacytometer).
5) Transfer 200,000 cells to a 15 mL conical tube and centrifuge at 1000 RPM for 4 minutes.
6) Resuspend the cell pellet in 4 mL of BME (5mg/mL) (prepared in step 4). Final cell stock will be 50,000 cells/mL. Keep on ice at all times.
7) Using a multi-channel pipette and reverse pipetting technique, seed cells (20 µL per well, 1000 cells per well) into every well of the ClearView insert plate in the CoolBox (4 °C).
Note: Use a pre-cooled reservoir boat when seeding cells.
8) Centrifuge ClearView plate in a cooled centrifuge (set at 4°C) for three minutes at 50 x g.
9) Place the ClearView plate at 37°C on a pre-warmed CoolSink and allow the BME to polymerize for 30-60 minutes.
10) During incubation, prepare chemoattractants (e.g. 2-fold serial dilutions of 10% FBS in F12 medium + ITS). Assay medium alone (F12 + ITS) should be used as a control.
11) Using a manual multi-channel pipette, add 200 μL of the chemoattractants and control assay medium to the appropriate wells of the second reservoir plate.
12) Using a manual multi-channel pipette, gently add 40 µL of assay medium to all insert wells on top of the BME:cell layer.
13) Carefully transfer the insert into the pre-loaded reservoir plate. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate.
14) 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.
15) In the IncuCyte® ZOOM software, schedule 24 hour repeat scanning (10x) every 2-3 hours. Typical assay duration is 72 hours.
Note: We recommend plotting bottom side fluorescent nuclear count as the assay metric
Figure 1. Determination of NucLight Red HT-1080 invasive and migratory response toward FBS. Plate map and corresponding microplate graph for FBS agonist curves. Serum starved NucLight Red HT-1080 cells were either embedded in 5mg/mL BME (invasion) or seeded directly onto the ClearView Cell migration plate membrane (migration control) at 1,000 cells per insert well. The indicated concentration of FBS was added to the reservoir plate and data were collected for 96 hours at 2 hour intervals. Each well is individually graphed illustrating the difference in kinetic movement of migrating and invading cells.
Figure 2. Representative morphological images of f HT-1080 cells. Migration: Images A, B, and C show the time-lapse progression of HT-1080s cells migrating toward 10% FBS. HT 1080 cells have a flat appearance and migration to the bottom side of the membrane (cells that are out of focus) can be seen prior to 24 hrs. Invasion Images D, E, and F show HT-1080 cells invading through the BME (5 mg/mL) toward the membrane pores. Cells have an elongated morphology, sending out laemellipodia or extensions to drive movement through a dense gel layer.