As the Production Team Leader at ArunA Biomedical, my duties include addressing the technical needs of customers. Some questions that consistently come up regard plate coating. When plating ArunA's hNP1™ Human Neural Progenitor Cells, and hN2™ cells, we recommend the use of Matrigel™ (BD Biosciences Cat #354234). For some laboratories, using Matrigel successfully can be tricky. To alleviate some of the frustration, I have included a list of helpful tips for first time users to become accustomed to working with the product. These are the steps we use in the lab at ArunA to make 1:200 Matrigel™ dilution, which we then use to coat our plates:
- It is extremely important to keep the Matrigel™, and all components used to make the solution cold/on ice.
- Thaw the stock bottle of Matrigel™ overnight at 4C. (You may also thaw it on ice while in the refrigerator; the ice helps to buffer temperature fluctuations and keep the Matrigel™ cold.)
- While on ice, dilute the thawed Matrigel™ with an equal volume of cold DMEM (e.g., mix 10 ml thawed Matrigel™ with 10 ml DMEM)- this makes a 1:2 dilution. We typically make 1-3ml aliquots. Immediately put the aliquots in the -20C.
- Before use, thaw a 1:2 Matrigel™ aliquot overnight at 4C until fully thawed (approximately 24hrs).
- Use the 1:2 dilution to further dilute to 1:200 (e.g., to make 100 ml solution, add 1ml of 1:2 Matrigel™ into 99 ml of DMEM).
- Coat the plates you will use fresh each time and let the Matrigel™ coated plate incubate at 4C or room temp in the hood for 1-3 hours.
- Be sure to wash once the PBS containing calcium and magnesium.
- Before using, you may want to consider checking the plate under the microscope for debris (see reference below). Never use a prepared Matrigel™ solution for longer than 2 weeks
Left, Poor Matrigel coating (note the high level of debris on the plate, little to no cell attachment).
Right, Successful Matrigel coating (little to no debris on the plate, high level of cell attachment).