PROTOCOL

Detailed Non-Adherent Chemotaxis Cell Migration Protocol


Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte® Chemotaxis Cell Migration Assay. Note, that the protocol does not include a description of any experiments required for optimizing seeding density or surface coatings. Here we specifically describe the use of the IncuCyte® ZOOM instrument for establishing and quantifying a chemotactic response of neutrophils toward fMLP.

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Neutrophil Chemotaxis

Materials

  • Freshly isolated neutrophils
  • RPMI-1640 + 0.5% Human Serum Albumin (Note: BSA CANNOT be used in replacement of HSA. We have found that neutrophils will not migrate toward C5a or IL-8 if BSA is used as the serum albumin)
    • RPMI-1640 (ATCC 30-2001)
    • HSA (Sigma-Aldrich A9731-5G)
  • IncuCyte® ClearView 96-Well Cell Migration Plate (Essen 4582 or 4599)
  • 2x IncuCyte® ClearView 96-Well Cell Migration Reservoir Plate (Essen 4600 or 4601)
  • Matrigel® Matrix (Corning 354234)
  • Fetal Bovine Serum (Sigma-Aldrich F2442-500mL)
  • D-PBS (w/o Ca2+, Mg2+, Life Technologies 10010)
  • fMLP (Sigma-Aldrich F3506-10MG)


Protocol

NOTE: All steps, other than the initial polymerization of the coating matrix, are performed at ambient temperature and using standard sterile cell culture technique prior to placing the cell migration plate into the IncuCyte® ZOOM instrument for imaging.

  1. Coat both sides of the membrane with 50 µg/mL Matrigel® + 10% FBS adding 20 µL to the insert wells (reverse pipette) and 150 µL to the reservoir wells (pre-fill reservoir and gently place the insert into the reservoir plate containing coating matrix). In this case, a second reservoir plate will be loaded with chemoattractant and used during the experiment. Learn how to coat

    NOTE: The cell migration plate and all reagents, must be pre-chilled to 4°C. We recommend using a CoolSink™ to keep the plate cold during the coating procedure.

  2. Place the ClearView Cell Migration plate at 37° C and incubate for 30 minutes.
  3. Remove the ClearView plate from 37° C and allow to cool down to ambient temperature for 30 minutes.

    NOTE: This step is important in order to achieve uniform cell distribution within each well.

  4. 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 83,333 cells per mL.

    NOTE: When studying inhibitors of cell migration, cell seeding volume is reduced to 40 µL per well, and the cell seeding stock is increased to 125,000 cells per mL. This allows for 20 µL addition of test reagent.

  5. Immediately prior to neutrophil addition, aspirate Matrigel® Matrix coating from both the reservoir plate and insert. Add 200 µL D-PBS to the reservoir plate then gently place the insert into the reservoir.
  6. Using a manual multi-channel pipette and reverse pipetting technique, seed cells (60 µL per well, 5,000 cells per well) into every well of the insert plate. Calculation: 83,333 cells/mL x 0.06 mL = 5,000 cells per insert well.
    Learn how to seed
  7. In order to eliminate bubbles that may be present on the membrane surface, after dispensing cells, use a manual multi- channel pipette and set at 40 µL and triturate wells. Learn how to remove bubbles
  8. Allow the neutrophils to settle at ambient temperature on a level surface for 45 – 60 minutes.
  9. During cell settling, prepare fMLP chemoattractant dilutions.
  10. Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir Plate.
  11. Carefully transfer the insert plate containing cells into the pre-filled second reservoir plate containing medium ± chemoattractant. Learn how to transfer
  12. Place the IncuCyte® 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.
  13. In the IncuCyte® ZOOM software; schedule 24 hour repeat scanning (10x) for every 30 minutes. NOTE: This schedule is only for scanning a single plate. Fewer scans will be required if scheduling multiple plates.
    • a) Objective: Ensure 10x objective is installed.
    • b) Vessel Type: Select "ClearView Cell Migration"
    • c) Channel Selection: Select "Phase"
    • d) Scan Mode: Select "Chemotaxis (Top/Bot)" scan type and desired Scan Pattern
    • Note: IncuCyte® instrument estimates a scan time of 20 minutes per plate (phase only); however, the actual scan time can take longer if condensation is not wiped away.

Example of Data Generated with the Detailed Non-Adherent Chemotaxis Cell Migration Protocol

Representative images of neutrophils migrating toward 50 nM fMLP

Representative images of neutrophils migrating toward 50 nM fMLP. A top-side segmentation mask (yellow) is blended with original phase contrast images of freshly isolated neutrophils migrating toward 50 nM fMLP taken at t=0, 2 hours, and 4 hours. Cells were seeded at 5,000 cells per well into an IncuCyte® ClearView 96-Well Cell Migration Plate.

Kinetic analysis of neutrophils migrating toward fMLP

Kinetic analysis of neutrophils migrating toward fMLP. Freshly isolated neutrophils were seeded on a coated ClearView insert (50 μg/mL Matrigel® Matrix + 10% FBS) and allowed to settle at ambient temperature for 1 hour. The insert was then placed into a reservoir containing decreasing dilutions of the fMLP. Total neutrophil area normalized to max area is plotted (n=4 per condition).

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