Matrigel Transwell/Boyden Chamber Invasion Assay

Reagents and chemicals

• Matrigel or basement membrane matrix is used as the ECM barrier on the porous insert membrane^[1][2][4].

• Serum-containing medium or another defined chemoattractant is placed in the lower chamber to generate a directional migration cue^[1][2].

• PBS is used for washing, and paraformaldehyde can be used for fixation before staining and image acquisition^[2].

Antibodies, probes, dyes, or kits

• Hoechst staining, crystal violet, or hematoxylin can be used to visualize invaded cells after fixation^[2][5].

• Matrigel composition can influence interpretation because regular and growth-factor-reduced Matrigel contain different matrix-associated proteins, proteases, inhibitors, and growth-factor content^[4].

Equipment and instruments

• The assay uses a Transwell or Boyden chamber insert with a porous membrane separating upper and lower medium compartments^[1][3].

• A fluorescence or brightfield microscope is used to image invaded cells, and ImageJ/Fiji or absorbance-based plate-reader workflows can be used for quantification depending on the staining method^[2][5][6].

Preparation Steps

• Prepare healthy cells in the appropriate growth medium before assay setup, because cell viability and baseline motility influence invasion readout^[2][3].

• For Matrigel coating, one published protocol thawed Matrigel at 4°C overnight, diluted Matrigel 1:3 in cold serum-free DMEM, coated the underside of the insert membrane with 25 µL Matrigel solution, and gelled the coating at 37°C for 30 min^[2].

• The same protocol used 8 µm pore membranes as an example, but pore size should be selected according to cell size^[2].

Operation Steps

• Place culture medium with or without chemoattractant in the lower chamber; one published protocol used 500 µL medium in the lower well of a 24-well plate and 10% FBS as the melanoma-cell chemoattractant condition^[2].

• Transfer the coated insert into the lower well, add the cell suspension to the upper chamber, and incubate at 37°C with 5% CO₂ for 20–24 h in the reported protocol^[2].

• After incubation, fix cells with 4% paraformaldehyde for 15 min at room temperature, wash both membrane sides twice with PBS, stain nuclei with Hoechst at 10 µg/mL for 15 min or use crystal violet or hematoxylin, then remove non-invaded cells from the upper membrane surface with gentle cotton-swab scraping before final imaging^[2].

Data Acquisition and Analysis

• Acquire images covering the membrane surface before and after removal of non-invaded cells when calculating the percentage of invaded cells, or count stained cells on the underside of the membrane as the endpoint readout^[2][3].

• Quantification may be performed by manual counting, ImageJ/Fiji-based analysis, imaging cytometry, dye extraction followed by absorbance measurement, or impedance-based real-time readout, but the same readout should be applied consistently within an experiment^[2][5][6].

• Include uncoated migration inserts, no-chemoattractant controls, vehicle controls, positive invasive-cell controls, and cell-viability controls when testing drugs or genetic perturbations, because reduced invasion can reflect either impaired invasion or reduced survival/proliferation^[3][6].

Problem: Matrigel coating is inconsistent or absent after setup.

• Possible Cause: Matrigel gels rapidly at room temperature and remains liquid at 4°C.
• Literature-supported Solution: Thaw Matrigel at 4°C overnight, keep materials cold during dilution and coating, and gel the coated insert at 37°C for at least 30 min^[2].

Problem: Invaded-cell counts are inaccurate.

• Possible Cause: Non-invaded cells remaining on the upper membrane surface can be counted as invaded cells.
• Literature-supported Solution: Remove upper-surface cells gently with a cotton swab before final imaging and quantify only cells remaining on the invaded side of the membrane^[2][3].

Problem: Apparent invasion differs between Matrigel lots or matrix types.

• Possible Cause: Matrigel and growth-factor-reduced Matrigel contain different bioactive components, including matrix enzymes, protease inhibitors, and growth factors.
• Literature-supported Solution: Record the Matrigel type, keep matrix type consistent within experiments, and interpret invasion changes in light of matrix composition^[4].