A neutral protease or cell recovery solution is recommended for harvesting cells cultured in GelNest™ Basement Membrane Matrix. Compared with trypsin, collagenase, or other proteolytic enzymes, a neutral protease can generate a single-cell suspension more gently and effectively without damaging the cells or cell-surface proteins. It is therefore suitable for cells that will be replated or analyzed. Neutral protease may also be used for tissue dissociation.

For metabolic studies and RNA extraction, use a cell recovery solution at 4°C for non-enzymatic cell collection. Because GelNest™ Basement Membrane Matrix contains trace amounts of RNA, include a matrix-only control without cells when performing RNA analysis. Another recovery method is to lower the temperature to 4–6°C to depolymerize the matrix; however, this requires time and is suitable only for certain applications.

Coated culture dishes should preferably be used on the same day, although the appropriate period depends on the experimental objective. If storage is necessary, they may be kept in a 37°C incubator for up to 7 days. During storage, evenly cover the GelNest™ Basement Membrane Matrix surface with serum-free medium to keep it moist.

A thin-gel coating is mainly used to promote cell attachment and proliferation. For example, primary cell culture may require only a thin protein layer to support attachment. A thick gel is mainly used for 3D cell culture, such as differentiation of rat aortic tissue into capillary-like structures in a ring assay, and for cell invasion assays. 3D cell culture is primarily used to study cell-cell interactions and complex structures such as biological tissues.

For this assay, the concentration of GelNest™ Basement Membrane Matrix should be at least 10 mg/mL.

Different experimental objectives require different concentrations of GelNest™ Basement Membrane Matrix, and users should determine the concentration according to their specific needs. The minimum gelation concentration for a thick-gel experiment is 7 mg/mL. Do not dilute solely by a fixed volume ratio, because protein concentration varies among lots. Calculate the required volume of diluent, such as PBS or serum-free medium, according to the desired final working concentration in mg/mL. For in vivo studies, the final working concentration should not be below 10 mg/mL to avoid incomplete gelation.