In the invasion experiment, cells should be added on the top layer of the matrix gel. Many literatures will directly lay out cells that have been treated with drugs in advance to perform cell invasion experiments. If you also want to add small molecule drugs, it is recommended that you refer to the literature to design the experiment.

Note: The matrix gel needs to be taken out from -20°C, thawed overnight at 4°C, kept on ice, diluted with ice-cold water (1:1 to 1:2), and then added to the Transwell insert and incubated at 37°C for 30 minutes to 1 hour to solidify. The specific operation is as follows (PMID: 32575572; PMID: 35840633; PMID: 37142933):

I. Experimental preparation

1. Move the Matrigel (extracellular matrix) from the -20°C refrigerator to the 4°C refrigerator overnight 1 day in advance to thaw (avoid repeated freezing and thawing).

2. On the day of the experiment, keep Matrigel on ice, dilute it with pre-cooled sterile deionized water at a ratio of 1:1 (e.g. 100 μL Matrigel + 100 μL deionized water), and mix gently (avoid bubbles).

3. Cells need to be cultured to 80-90% confluence, digested with 0.25% trypsin-EDTA, centrifuged at 335×g in a 15mL centrifuge tube for 5 minutes, and discard the supernatant. Wash the cells twice with PBS (discard the supernatant after each centrifugation), and finally resuspend them in migration buffer (DMEM + 10mM HEPES + 0.1% BSA, pH 7.4), count with trypan blue, and adjust the cell concentration to 1×10⁶ cells/mL.

II. Operation steps

1. Matrigel plating

In a clean bench, gently add 50-100 μL of diluted Matrigel to the Transwell insert (8 μm pore size) membrane of a 24-well plate, ensuring that the Matrigel evenly covers the membrane surface (avoid bubbles).

Place the Transwell plate in a 37°C, 5% CO₂ incubator for 30 minutes to 1 hour to allow the Matrigel to solidify into a gel.

2. Drug addition and cell inoculation

If the drug is tested for its inhibitory effect on cell invasion, add the drug (PMID: 32575572) to the cell suspension and gently mix (the drug needs to be diluted with migration buffer in advance). Carefully add 100 μL of drug-containing cell suspension (about 1×10⁵ cells/well) to the solidified Matrigel, avoiding touching the Matrigel or membrane surface.

Return the Transwell plate to the incubator and incubate for 10 min to allow the cells to settle on the surface of the Matrigel.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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Cell invasion assays using basement membrane matrix gels (e.g., Matrigel® or collagen-based gels) model cellular migration through a simulated extracellular matrix (ECM) to study metastatic potential, angiogenesis, or immune cell infiltration. The assay typically employs a Boyden chamber (Transwell®) or 3D spheroid setup, where cells penetrate a gel layer (like Matrigel, a laminin/collagen-rich matrix) to reach a chemoattractant. Key steps include careful gel preparation—thawing Matrigel on ice, diluting it in serum-free medium (1–2 mg/mL), and polymerizing it on Transwell inserts (50–100 µL, 37°C for 30–60 min). Cells are serum-starved (6–24 hrs) to minimize baseline motility, seeded in the upper chamber, and allowed to invade toward a chemoattractant (e.g., 10% FBS or EGF) in the lower chamber for 24–72 hrs. Post-incubation, invaded cells are fixed (methanol/formalin), stained (crystal violet or calcein-AM), and quantified via microscopy or fluorescence.

Critical optimization includes adjusting gel density (higher concentrations increase stringency), cell numbers (10⁴–10⁵ cells/insert), and controls (invasive vs. non-invasive cell lines, chemoattractant omission). Pitfalls like uneven gel coating, high background (mitigated by swabbing non-invaded cells), or false positives (verified via migration-only controls) require attention. Advanced adaptations include 3D spheroid embedding, live-cell imaging (FITC-labeled gels), or drug testing (e.g., MMP inhibitors). Alternatives to Matrigel include collagen I/IV (more defined but less bioactive), synthetic PEG hydrogels, or decellularized tissue-specific ECM. Data should normalize to migration controls, viability assays, and matrix degradation metrics (e.g., MMP zymography). For tailored protocols, specifying cell type and research goals can refine experimental design.