Application of OrganoGel in Transwell Cell Invasion Assays In Vitro

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I. Introduction to Matrigel

Matrigel is a basement membrane extract derived from mouse tumor tissue (the basement membrane is a matrix membrane on the basal surface of epithelial cells within animals), which forms the Matrigel (Figure 1). The main components of Matrigel are laminin, type IV collagen, and nidogen. Additionally, Matrigel also contains a variety of growth factors, such as Epidermal Growth Factor (EGF), Platelet-Derived Growth Factor (PDGF), Nerve Growth Factor (NGF), Basic Fibroblast Growth Factor (FGF-2), Transforming Growth Factor-beta (TGF-beta), and Insulin-Like Growth Factor (IGF) (Vukicevic et al. 1992).

Figure 1: Distribution of the basement membrane in mouse tumor tissue.

I. Transwell Cell Invasion Assay In Vitro

The Transwell Cell Invasion Assay In Vitro is applied to study the effects of various cytokines on the invasion and metastasis of malignant tumor cells, as well as the research of new drugs that inhibit angiogenesis. The principle of the Transwell experiment involves placing the Transwell chamber into a culture plate and separating high-nutrient medium from low-nutrient medium with a membrane. Cells are placed in the low-nutrient medium, and in order to obtain more nutrients, they will migrate through the membrane into the high-nutrient medium. The Transwell cell experiment can serve as a very simple method for studying the migration, invasion, and metastasis of tumor cells.

Transwell Experiment Principle Diagram

2.1 Experimental Materials

NO.	Materials	Notes
1	Transwell Culture Chamber
2	Matrigel
3	Cell Culture Plate
4	Supernatant Medium	Serum-free Medium Supplemented with 0.05%-0.2% BSA
5	Underlying Medium	Culture Medium Containing 5%-10% FBS

2.2 Experimental Method
1. Coating with Matrigel: Dilute the Matrigel with serum-free cell culture medium or PBS buffer at a ratio of 1:8 at 4°C (the dilution ratio should be determined experimentally, choosing a concentration that allows cells to pass through moderately is sufficient). Take 100 µL and evenly spread it onto the surface of the polycarbonate membrane in the upper chamber. Place it at 37°C for 0.5-1 hour to allow it to polymerize into a gel.

Note: (a) Gently dispense the Matrigel along the inner wall of the chamber, avoiding puncturing the filter membrane; (b) The volume of Matrigel added should not be too large. It should be just enough to wet the polycarbonate membrane; (c) Pay attention to low temperatures, and pre-cool the pipette tips, chambers, etc., at 4°C.

2. Cell culture: Take cells in the logarithmic growth phase and wash them with PBS. Then, suspend the cells in serum-free culture medium and adjust the cell density to 1-10 * 10^5/ml.

3. Seeding cells: Generally, add 500-650 µL of culture medium containing 5%-10% FBS or chemotactic factors to the lower chamber of a 24-well plate. Place the Transwell chamber in the 24-well plate using tweezers, take 100-200 µL of cell suspension and add it to the upper chamber, and finally place it in the incubator for 12-48 hours (depending on the metastatic ability of the cancer cells).

Note: (a) Try to avoid the formation of bubbles: Bubbles often form between the lower culture medium and the chamber, which can affect the chemotactic effect of the lower culture medium. Therefore, if a large bubble appears, lift the chamber, remove the bubble, and then place the chamber back into the culture plate; (b) Ensure that the cells are seeded evenly, and it is recommended to add them slowly along the wall. (c) The choice of time points should not only consider the cell's metastatic ability but also the impact of treatment factors on cell numbers, such as certain drugs that may inhibit cell proliferation.

4. Cell fixation: Remove the chamber, aspirate the culture medium, and gently wipe the Matrigel and cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 600 µL of 4% paraformaldehyde, place the chamber in it, and fix for 20-30 minutes.

5. Cell staining and counting: After rinsing with the fixing solution, stain with 0.1-0.2% crystal violet for 5-10 minutes, wash three times with PBS to remove unbound crystal violet, gently wipe the upper side of the chamber with a cotton swab to remove the dye that is non-specifically bound to the upper surface of the chamber, in preparation for subsequent microscopy. After air-drying appropriately, observe and count the cells in 5 fields of view under a high-power microscope.

Recommended cell seeding numbers per well for common cell types (for reference only)

Cell Name	Cell Seeding Density	Detection Time
DU145	3*10⁴	24h
PC3	5*10⁴	24h
A549	8*10⁴	48h
22RV1	8*10⁴	72h

Item NO.	Product Name	Size
abs9410	Ready to use Matrigel	100ml
abs9490	Standard OrganoGel with Phenol red	1.5ml*8
abs9491	Standard OrganoGel Phenol red free	1.5ml*8
abs9492	HC OrganoGel with Phenol red	1.5ml*8
abs9493	HC OrganoGel Phenol red free	1.5ml*8
abs9494	GFR OrganoGel with Phenol red	1.5ml*8
abs9495	GFR OrganoGel Phenol red free	1.5ml*8
abs9496	IPS-qualified OrganoGel Phenol red free	1.5ml*4
abs9497	HC&GFR OrganoGel with Phenol red	1.5ml*8
abs9498	HC&GFR OrganoGel Phenol red free	1.5ml*8

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Absin Bioscience Inc.
Email: worldwide@absin.cn

September 11, 2024

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