PROTOCOL

IncuCyte® Chemotaxis Cell Migration General Protocol


IncuCyte® Chemotaxis Cell Migration General Protocol

This protocol provides an overview of the IncuCyte® Chemotaxis Cell Migration Assay methodology, which can be used with adherent or non-adherent cell types. This protocol is compatible with the IncuCyte® instrument using nuclear labeled or non-labeled cells.

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  1. Seed cells of interest at 60 µL per well (40 µL per well if adding inhibitors of cell motility; refer to step 2) at an appropriate density into a ClearView 96-well insert. Typically this is done with the insert mated to an empty reservoir plate. The seeding density will need to be optimized for each cell type used, however, we have found that 1,000 cells per well for adherent cell types and 5,000 cells per well for non-adherent cell types are reasonable starting points. Learn how to seed cells
    • Some cell types may require reduced exposure to Fetal Bovine Serum (FBS) prior to initiating this transmembrane assay (e.g. HT-1080s starved in F12 + Insulin-Transferrin-Selenium for ~20 hours).
    • Some cell lines (e.g. neutrophils) may require the addition of a basement membrane extract (e.g. 50 µg/mL Matrigel® + 10% FBS) to promote light cell adherence and provide the necessary integrins for cell motility. Follow manufacturer’s recommendations for coating. Refer to Table 1 for cell line seeding density and coating recommendations. Learn how to coat
  2. If pre-treating cells, prepare 3x treatments and immediately add 20 µL to the insert wells containing cells immediately after cell seeding. Triturate the cells, using a 30 µL volume, to appropriately mix the treatment, so cell exposure during pre-treatment is at 1X.

    NOTE: It is important to immediately add treatment to insert wells immediately after seeding to avoid cell settling prior to treatment addition. If adherent cells attach prior to trituration, uneven cell distribution can occur.

  3. Place the plate onto a level surface and allow the cells to settle at ambient temperature for 15 minutes (adherent cell types) to 60 minutes (non-adherent cell types). For adherent cell types, we recommend a continued pre-incubation with inhibitors at 37°C for 30 minutes.
  4. Add 200 µL of desired chemoattractant or control to the appropriate wells of a second reservoir plate. Carefully transfer the insert (Learn how to 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. Learn how to remove bubbles
  5. 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, remember to carefully wipe away any condensation that may have accumulated on the plate lid or bottom of the reservoir.
  6. Image the plate in the IncuCyte® ZOOM instrument with a 10x objective using the Chemotaxis Scan Type.

Table 1. Validated Cell Lines and Recommendations

Validated Cell Lines and Recommendations

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