Standard Operating Procedure (SOP) for Transwell Migration Assay

Disclaimer

This SOP provides a general guideline for conducting cell migration assays using transwell chambers. It is crucial to note that specific parameters, including seeding density, incubation times, and concentrations of chemoattractants or inhibitors, may vary depending on the cell line and experimental objectives. Researchers are advised to optimize these parameters for their specific cell lines and experimental designs before proceeding.

Background

Cell migration is a process that plays a crucial role in various physiological and pathological conditions, including wound healing, immune response, and cancer metastasis. Understanding cell migration mechanisms can provide insights into the development and progression of diseases, as well as contribute to the development of novel therapeutic strategies. Migration assays, such as the transwell migration assay, are crucial in studying the motility of cells in vitro. These assays allow researchers to quantify the movement of cells through a semipermeable membrane towards a chemoattractant, mimicking the migration of cells in vivo. By manipulating the conditions under which these cells migrate, including the use of inducers or inhibitors that can either promote or inhibit migration, scientists can uncover the molecular pathways involved in cell movement and identify potential targets for therapy.

Objective

To measure the migratory response of cells to a chemoattractant or the effect of a migration inhibitor or inducer using a Transwell migration assay.

Materials

• 24-well plates
• 8 µm Transwell® with 8.0 µm pore polycarbonate membrane insert (e.g., Greiner Bio-One)
• Cell culture medium with low serum concentration (e.g., 0.5% FBS)
• Chemoattractant of choice
• Migration inhibitor or inducer (if applicable)
• 4% Paraformaldehyde (PFA)
• 0.1% Crystal violet solution
• 90% Acetic acid
• Cotton swabs
• Microscope
• Microplate reader capable of reading absorbance at 590 nm

Procedure

1. Chemoattractant Addition: Fill the lower compartment of the well with 600 µL of medium containing the chemoattractant (inducer). If your chemoattractant is an inhibitor, you will resuspend it with the medium and the cells to evaluate whether the inhibitor affects the ability of the cells to move through the membrane (see steps 2 and 3).
2. Preparation of Cell Suspensions: Resuspend cells in a low serum medium (e.g., 0.5% FBS) to a suitable concentration (e.g., 1×10^5 cells/mL).
3. Seeding of Cells: Add 200 µL of the cell suspension to the upper compartment of the insert placed in a 24-well plate.
4. Incubation: Incubate the plates for 24 hours (or appropriate time based on cell type and experimental needs) at 37 °C in a 5% CO2 atmosphere to allow cell migration through the pores.
5. Fixation and Staining: After incubation, carefully remove the non-migrated cells from the upper surface of the membrane with a cotton swab. Fix migrated cells on the lower surface with 4% PFA for 15 min at room temperature. Wash twice with 1xPBS and then stain with 0.1% crystal violet for 30 min.
6. Washing: Wash the membranes gently with 1xPBS to remove excess stain.
7. Microscopic Imaging: After washing, take images of the stained migrated cells under the microscope for documentation and qualitative analysis.
8. Dye Elution and Quantification: Elute the dye from the migrated cells with 90% acetic acid. Measure the absorbance of the eluted dye solution at 590 nm using a microplate reader. The absorbance is directly proportional to the number of migrated cells.

Data Analysis

Analyze the data by comparing the absorbance values from wells with chemoattractant alone to those with chemoattractant and migration inducer or inhibitor. Use control wells with cells and low serum medium without chemoattractant for background correction.

The schematic illustration included in this SOP is a visual representation of the transwell migration assay process and was generated using BioRender. This diagram is intended to provide a clear and concise overview of the experimental setup and procedural steps.