Cells used in the experiment should be no later than passage 12 and should be passaged at least twice after recovery from liquid nitrogen before use.

Ensure that the matrix surface remains even and level during gelation, and use a culture plate with a larger surface area whenever possible.

Cell viability should be greater than 95% at the time of seeding.

When adding cells to the wells, mix them evenly because cell density affects tube formation.

Matrix concentration: the stock concentration should be at least 10 mg/mL, and the final diluted concentration should be above 6 mg/mL. The optimal concentration for tube formation is 6–8 mg/mL.

Remove the 5 mL bottle of GelNest™ Matrix and place it in an ice-water bath. Use crushed ice and pack it firmly. Insert the bottle upright, then add water until the water level is above the matrix level, while keeping the bottle cap out of the water.

Place the ice-water bath containing the matrix in a 4°C refrigerator overnight, for approximately 16 hours, to achieve complete liquefaction.

Once the matrix has fully liquefied, mix gently by pipetting, aliquot it, and store the aliquots at -20°C for long-term storage.

Prepare a box of sterile 1,000 μL pipette tips and the required number of sterile 1.5 mL microcentrifuge tubes, and place them in an ice-free cooling box or tube rack.

Pre-cool all consumables in a -20°C freezer for at least 1 hour before aliquoting to prevent the matrix from warming and gelling prematurely.

Mix the matrix thoroughly before aliquoting. Fully liquefied GelNest™ Matrix should flow freely and contain no gel clumps.

Dispense the mixed matrix into 1.5 mL microcentrifuge tubes.

Store the aliquots at -20°C for up to 6 months or at -80°C for long-term storage.

Remove the aliquoted GelNest™ Matrix from the -20°C freezer.

Place it in an ice-water bath or a 0°C metal block. After approximately 30 minutes, the matrix in the microcentrifuge tube should thaw from -20°C to a liquid state.

GelNest™ Basement Membrane Matrix is a reconstituted basement membrane extracted from mouse tumors. The freshly extracted material mainly contains laminin (60%), type IV collagen (30%), entactin (8%), heparan sulfate proteoglycan (2%), epidermal growth factor, insulin-like growth factor, and other growth factors.

At low temperatures, such as 4°C, interactions between protein molecules are weak. Insufficient energy prevents the formation of intermolecular noncovalent bonds, leaving the molecules relatively free. GelNest™ Basement Membrane Matrix therefore remains fluid and can be pipetted or mixed with cells.

At 8–37°C, rising temperature changes protein conformation, strengthens hydrophobic interactions, and reduces intermolecular distance. Van der Waals forces, hydrogen bonds, and electrostatic interactions stabilize intermolecular associations. Entactin acts as a cross-linking molecule between laminin and type IV collagen, together promoting formation of a stable basement membrane structure and causing GelNest™ Basement Membrane Matrix to gel.