DendroTEK dPGA
Adding Laminin to as a secondary layer
Version 1.0 | For Research Use Only
Introduction
Laminin is a key extracellular matrix (ECM) protein found in the basal lamina of epithelial and endothelial tissues. In vitro, it supports the adhesion, survival, differentiation, and morphological development of diverse cell types, including neural, hepatic, endothelial, epithelial, and muscle cells.
This protocol describes how to apply laminin as a secondary coating following an initial substrate such as Poly-L-Ornithine (PLO) or DendroTEK dPGA.
Materials Required
● Laminin (murine, EHS-derived) – Sigma Aldrich #L2020 or Recombinant CellAdhere™
Laminin-521 (STEMCELL Technologies, #77003)
● Sterile 1X PBS (Ca²⁺/Mg²⁺ free)
● Sterile ddH₂O
● 0.22 μm Teflon filter
● Sterile cultureware
● Ice and ice bucket
Storage and Stability
● Laminin: Store undiluted at –20 °C, avoid freeze-thaw cycles.
○ Do not let laminin gel — irreversible denaturation occurs upon warming.
Stock Preparation
Laminin Working Aliquots
1. Thaw the laminin stock slowly on ice to avoid gelling.
2. Optional: Prepare single-use aliquots (e.g., 10–50 µL) in sterile PBS or ddH₂O.
3. Store aliquots at –20 °C until needed.
Primary Coating with dPGA
See dPGA Coating Protocol for detailed steps. Briefly:
● Coat with 50 µg/mL dPGA → Incubate 1 hr at 37 °C → Rinse 3× PBS.
Choosing Your Laminin
It is important to use a quality ECM after the primary coating. We recommend two ECMs
depending on your need.
● Laminin (Sigma, #L2020):
Standard option for general use. Works well with neurons, epithelial, and muscle cells.
Widely used, cost-effective.
● CellAdhere™ Laminin-521 (STEMCELL Technologies, #77003)
Recombinant human laminin. Best for human iPSCs and neural cultures. Xeno-free,
highly consistent.
Secondary Coating with Laminin
1. Thaw laminin slowly on ice (avoid warming to prevent gelling).
2. Dilute to working concentration in sterile PBS.
○ Typical starting concentration: 1 µg/mL
○ Some cell types may require 2–10 µg/mL for optimal adhesion.
3. Add to the coated plate surface. Ensure full coverage.
4. Incubate at 37 °C for 1 hour in a humidified incubator.
○ Optional: Incubate overnight at 4 °C for enhanced ECM adsorption.
5. After incubation, aspirate and wash 3× with sterile PBS.
6. Keep wells filled with PBS or media until seeding.
○ Do not allow the surface to dry for more than 2–3 minutes.
Assessing Success
Evaluate coating success via:
● Microscopy: Cells should adhere and spread uniformly within 6–24 hours.
● Adhesion test: Compare cell attachment vs. uncoated or single-coat controls.
References
● Clément, J.-P., et al. (2022). Dendritic Polyglycerol Amine: An Enhanced Substrate to
Support Long-Term Neural Cell Culture. ASN Neuro, 14, 17590914211073276.
https://doi.org/10.1177/17590914211073276