Matrigel/ECM Transwell Invasion Assay
Materials Required
Principle
The Matrigel/ECM Transwell invasion assay measures the ability of cells to move toward a chemoattractant while crossing an extracellular-matrix barrier placed on a porous membrane; therefore, the readout reflects both chemotactic motility and matrix invasion rather than migration alone[1][2][3]. Matrigel is a basement-membrane-rich matrix derived from Engelbreth-Holm-Swarm mouse sarcoma and has been used as a reconstituted basement membrane barrier in chemoinvasion assays[3][4]. The assay readout is generated by quantifying cells that reach the underside of the insert membrane or lower compartment after incubation, commonly by staining and counting invaded cells or by fluorescence-based quantification[1][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use Matrigel or another ECM coating to form the invasion barrier on the Transwell membrane; use serum-free or low-serum medium for the upper chamber and chemoattractant-containing medium, commonly serum-containing medium or defined factors, in the lower chamber[1][2][3].
• Use phosphate-buffered saline or appropriate culture medium for washing, fixation reagent for endpoint staining workflows, and crystal violet or DAPI when using microscopy-based cell counting[1][5].
• Crystal violet can be used to stain invaded cells on transparent membranes, while DAPI-based nuclear staining can improve counting on transparent inserts; Calcein-AM has been used for fluorescence-based invaded-cell quantification in FluoroBlok-style workflows[1][5][6].
• Use a multiwell plate with porous Transwell inserts, a humidified 37 °C and 5% CO2 incubator, sterile cell-culture equipment, a microscope for stained-cell counting, and a fluorescence plate reader or fluorescence microscope when using fluorescence-based readouts[1][5][6].
• Use phosphate-buffered saline or appropriate culture medium for washing, fixation reagent for endpoint staining workflows, and crystal violet or DAPI when using microscopy-based cell counting[1][5].
• Crystal violet can be used to stain invaded cells on transparent membranes, while DAPI-based nuclear staining can improve counting on transparent inserts; Calcein-AM has been used for fluorescence-based invaded-cell quantification in FluoroBlok-style workflows[1][5][6].
• Use a multiwell plate with porous Transwell inserts, a humidified 37 °C and 5% CO2 incubator, sterile cell-culture equipment, a microscope for stained-cell counting, and a fluorescence plate reader or fluorescence microscope when using fluorescence-based readouts[1][5][6].
Experimental Procedure
• Prepare healthy single-cell suspensions in the upper-chamber medium, prepare the lower-chamber chemoattractant medium, and keep Matrigel cold before coating because published Transwell invasion protocols place cells above an ECM layer and chemoattractant below the porous membrane[1][2].
• For self-coated inserts, published protocol descriptions report adding Matrigel to 24-well Transwell inserts and allowing it to solidify at 37 °C for 15-30 min; for pre-coated Matrigel invasion chambers, published use includes rehydrating inserts for 2 h before seeding cells[1][6].
• Select cell input empirically within the literature-reported range for the model system; examples include 25,000 MDA-MB-231 cells per 24-well Matrigel insert and 1-1.5 × 105 cells in BioCoat Matrigel chambers in a cancer-cell invasion workflow[6][7].
• Add chemoattractant medium to the lower chamber, place the ECM-coated insert into the well, seed cells into the upper chamber, and incubate under standard cell-culture conditions; published examples include 37 °C, 5% CO2, 6-8 h or 16 h imaging time points for MDA-MB-231 cells, 22-24 h for brain tumor stem-cell invasion workflows, and 48 h in some cancer-cell invasion assays[6][8][9].
• At endpoint, remove non-invaded cells from the upper surface when using fixed endpoint microscopy, then fix and stain invaded cells on the lower membrane surface or quantify fluorescently labeled invaded cells according to the chosen readout[1][5][9].
• For microscopy-based quantification, image defined fields or the full membrane area and count invaded cells; for fluorescence-based workflows, quantify signal from invaded labeled cells using the reported excitation/emission settings or microscope/plate-reader workflow used for the selected dye[5][9].
• Report invasion as invaded-cell number, fluorescence intensity, or normalized invasion relative to a control group; include a migration-only control without ECM when the experimental question requires separation of motility from ECM invasion[1][2][5].
• Use non-chemoattractant or serum-free lower-chamber conditions as a background control and chemoattractant-containing lower medium as the invasion condition; compare treatments only when cell viability or proliferation differences do not confound the invaded-cell readout[1][5].
• For transparent inserts, counting cells remaining on the upper side of the membrane can improve interpretation because Stoellinger and Alexanian reported that accounting for cells on top of the membrane improves accuracy of migration/invasion percentage estimation[5].
• For self-coated inserts, published protocol descriptions report adding Matrigel to 24-well Transwell inserts and allowing it to solidify at 37 °C for 15-30 min; for pre-coated Matrigel invasion chambers, published use includes rehydrating inserts for 2 h before seeding cells[1][6].
• Select cell input empirically within the literature-reported range for the model system; examples include 25,000 MDA-MB-231 cells per 24-well Matrigel insert and 1-1.5 × 105 cells in BioCoat Matrigel chambers in a cancer-cell invasion workflow[6][7].
• Add chemoattractant medium to the lower chamber, place the ECM-coated insert into the well, seed cells into the upper chamber, and incubate under standard cell-culture conditions; published examples include 37 °C, 5% CO2, 6-8 h or 16 h imaging time points for MDA-MB-231 cells, 22-24 h for brain tumor stem-cell invasion workflows, and 48 h in some cancer-cell invasion assays[6][8][9].
• At endpoint, remove non-invaded cells from the upper surface when using fixed endpoint microscopy, then fix and stain invaded cells on the lower membrane surface or quantify fluorescently labeled invaded cells according to the chosen readout[1][5][9].
• For microscopy-based quantification, image defined fields or the full membrane area and count invaded cells; for fluorescence-based workflows, quantify signal from invaded labeled cells using the reported excitation/emission settings or microscope/plate-reader workflow used for the selected dye[5][9].
• Report invasion as invaded-cell number, fluorescence intensity, or normalized invasion relative to a control group; include a migration-only control without ECM when the experimental question requires separation of motility from ECM invasion[1][2][5].
• Use non-chemoattractant or serum-free lower-chamber conditions as a background control and chemoattractant-containing lower medium as the invasion condition; compare treatments only when cell viability or proliferation differences do not confound the invaded-cell readout[1][5].
• For transparent inserts, counting cells remaining on the upper side of the membrane can improve interpretation because Stoellinger and Alexanian reported that accounting for cells on top of the membrane improves accuracy of migration/invasion percentage estimation[5].
Troubleshooting
Problem: Few or no invaded cells are detected.
• Possible cause: The assay duration, cell number, or chemoattractant condition may be unsuitable for the cell type.• Literature-supported solution: Optimize cell seeding density and incubation time within reported ranges for the cell model, because published workflows vary from 25,000 cells to 1-1.5 × 105 cells per insert and from 6-8 h to 48 h endpoints[6][7][9].
Problem: Invasion signal is difficult to interpret.
• Possible cause: Migration and invasion are being conflated.• Literature-supported solution: Include parallel uncoated migration inserts and ECM-coated invasion inserts because Transwell migration measures chemotaxis through pores, whereas Transwell invasion adds an ECM barrier[1][2].
Problem: Cell counting is inaccurate on transparent membranes.
• Possible cause: Manual counting may miss cells or confuse pores with nuclei.• Literature-supported solution: Use DAPI nuclear staining and overlay bright-field pore images with DAPI images, because this modification improved accuracy in a published Transwell migration/invasion method study[5].
References:
- [1]. Justus CR, et al. In vitro cell migration and invasion assays. J Vis Exp. 2014;(88):e51046. [Content Brief]
- [2]. Justus CR, et al. Transwell in vitro cell migration and invasion assays. Methods Mol Biol. 2023;2644:349-359. [Content Brief]
- [3]. Albini A, et al. The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation. Nat Protoc. 2007;2(3):504-511. [Content Brief]
- [4]. Passaniti A, et al. Matrigel: history/background, uses, and future applications. J Cell Commun Signal. 2022;16(4):621-626. [Content Brief]
- [5]. Stoellinger HM, et al. Modifications to the transwell migration/invasion assay method that eases assay performance and improves the accuracy. Assay Drug Dev Technol. 2022;20(2):75-82. [Content Brief]
- [6]. Renz M. In invasion assays, the breast cancer cell nucleus leads the way. BMC Res Notes. 2020;13(1):480. [Content Brief]
- [7]. Weitz AC, et al. Functional assay of cancer cell invasion potential based on mechanotransduction of focused ultrasound. Front Oncol. 2017;7:161. [Content Brief]
- [8]. Al-kharboosh R, et al. The study of brain tumor stem cell invasion. Methods Mol Biol. 2019;1869:111-121. [Content Brief]
- [9]. Wang C, et al. Evaluation of CD44 and CD133 as cancer stem cell markers for colorectal cancer. Oncol Rep. 2012;28(4):1301-1308. [Content Brief]
LinkedIn
Researchgate
Instagram
X
Facebook
WhatsApp
Reddit
Mail