IncuCyte® Chemotaxis Cell Invasion General Protocol

This protocol provides an overview of the IncuCyte® Chemotaxis Cell Invasion Assay methodology and is compatible with the IncuCyte® ZOOM instrument. Measurements of chemotactic cell invasion can be made using nuclear labeled or unlabeled cells, however, we recommend labeling cells with a live-cell nuclear label (e.g. IncuCyte® NucLight™ reagents) to ensure optimal performance.

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General Protocol

1. Pre-cool a ClearView Chemotaxis plate in a CoolBox system containing a frozen cold pack and CoolSink plate (4°C), for 5 minutes. Learn How to set up CoolBox™ accessories for IncuCyte®
   
   Note: It is important to keep close temperature control of biomatrix materials such as Collagen-1 and basement membrane extract (BME) to prevent unwanted gelling. 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.
   
   How to prime the ClearView membrane
   
   
3. While the ClearView plate is priming, prepare your biomatrix reagent at the desired working concentration as per the manufacturer's instructions.
   
   Note: Keep biomatrix reagents cool (4-8°C) by placing them in a CoolBox system or by embedding them in ice. Biomatrix solutions can be viscous. Prepare a large dead volume to ensure available biomatrix for transfer to the assay plate (e.g., prepare 4 mL biomatrix solution to provide 20 µL per insert well).
   
   
• The IncuCyte® Chemotaxis Cell Invasion assay is compatible with your choice of biomatrix gel. Recommended biomatrix proteins include: Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract (Trevigen 3445-005-01) and Cultrex® Rat Collagen I (Trevigen 3440-100-01).
• The required biomatrix density will be dependent on the matrix and cell types used. For HT-1080 cells we recommend BME (5 mg/mL) diluted in assay medium or Collagen I (1 mg/mL) diluted in neutralizing buffer (DMEM, Sigma D2429, + 7.5 g/L sodium bicarbonate + 0.004 g/L folic acid + 1% GlutaMax). When preparing collagen I it is important that it is properly neutralized to ensure cell health is maintained and gelling is uniform.
  
  
4. Harvest your cells and resuspend the pellet in the biomatrix solution. Cell density will need to be optimized for each cell type used; however, we have found that 1,000 cells per well is a reasonable starting point.
   
   How to resuspend your cells within a biomatrix
   
   Calculation: 50,000 cells/mL x 0.02 mL/well = 1,000 cells/well
   
   Note: Some cell types may require reduced exposure to Fetal Bovine Serum (FBS) before initiating the transmembrane invasion assay (e.g., HT-1080s starved in F12 + Insulin-Transferrin-Selenium for ~20 hours).
   
   
5. Seed cells (20 µL per well, 1,000 cells per well) into every well of the cooled ClearView insert plate.
   
   How to add a biomatrix cell suspension to the ClearView insert
   
   Note: Use a pre-cooled reservoir boat when seeding cells.
   
   
6. Centrifuge the ClearView plate in a cooled centrifuge (set at 4°C) for three minutes at 50 x g.
7. Place the ClearView plate at 37°C on a pre-warmed CoolSink and allow the biomatrix to polymerize for 30-60 minutes.
   
   How to polymerize your biomatrix
   
   
8. Gently add 40 µL of assay medium ± modulators of invasion on top of the biomatrix:cell layer in the insert wells.
   
   Note: If adding modulators of invasion, the working concentration should be 1.5x the desired final concentration to account for the volume of the biomatrix:cell layer.
   
   
9. Add 200 μL of desired chemoattractant, or control, to the appropriate wells of a second reservoir plate. Carefully transfer the insert into the pre-loaded reservoir plate. Learn how to transfer the insert. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate.
10. 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.
11. Image the plate in the IncuCyte® ZOOM instrument with a 10x objective, using the Chemotaxis Scan Type with “Phase” and the appropriate "Red" or "Green" channels selected dependent on the fluorescent label used.

Best Practices for Avoiding Bubbles

Air bubbles trapped on either side of the membrane can interfere with proper focusing and image processing. We recommend the following techniques to eliminate bubbles from your experiment:

• Reverse pipette during the biomatrix:cell addition step and when adding medium ± treatment on top of the polymerized biomatrix:cell layer. Reverse pipetting reduces the risk of splashing or bubble formation. In reverse pipetting, the volume aspirated into the tip is larger than the volume delivered to the receiving vessel:
  
  
1. Press the plunger to the second stop.
2. Dip the pipette-tip into the solution.
3. Release the plunger until the starting position has been reached.
4. Move the pipette-tip to the receiving vessel.
5. Dispense the liquid by pressing the plunger to the first stop. SOME LIQUID WILL REMAIN IN THE TIP.
6. Repeat steps 2–5 throughout the plate.
   
   
• Remove bubbles at the liquid surface by gently squeezing a wash bottle (containing 100% ethanol with the inner straw removed) to blow vapor over the surface of each well.
• When replacing the ClearView insert into the pre-filled reservoir do so at a slight angle to ensure the reservoir liquid displaces all air trapped under the membrane.