Stem Cell Qualified ECM Gel Coating ProtocolNOTE: Expansion of human pluripotent stem cells requires culture ware that is coated with prequalified ECM proteins. Below are general guidelines for the coating of 6-well plates using the Stem Cell Qualified ECM Gel (CC131). All procedure should be performed under aseptic conditions in a biological safety cabinet.1. Thaw the Stem Cell Qualified ECM Gel in the 2-8°C fridge one day before use. Once thawed, maintain ECM Gel on ice at all times and use pre-cooled medium and pipettes to avoid gelling of the product. IMPORTANT: Do not thaw ECM Gel at temperatures higher than 15ºC to avoid gelling. 2. Dilute the ECM Gel 1:80 with cold DMEM/F12 (D6421) medium. Scale up or down according to the volumes required. Below is an example for coating a 6-well plate with 1.5 mL of 1:80 diluted ECM Gel.• To make a 1:20 dilution, add 0.5 mL ECM Gel to 9.5 mL cold DMEM/F12 (D6421) or DMEM (D5796) medium into a 15 mL conical tube. Total volume = 10 mL. • A further 4-fold dilution is required to make the final 1:80 dilution. Add 2.5 mL of 1:20 diluted ECM Gel to 7.5 mL cold DMEM/F12 medium. Total volume = 10 mLNOTE: The recommended dilution is 1:80, however more concentrated Stem Cell Qualified ECM Gel may be used if desired.3. Add 1.5 mL of the 1:80 diluted Stem Cell Qualified ECM Gel to each well of a 6-well plate. Swirl the culture plates to spread the ECM Gel evenly across the surface of the plate. Store in a 2 -8°C fridge overnight or at least 2 hours in the fridge before use. If not used immediately, parafilm wrap the ECM coated culture plates and store at 2-8°C until ready to use. Use the ECM coated culture plates within 3-4 days.4. Prior to seeding the cells, bring the plate back to room temperature for 10-15 minutes, remove the coating solution and add 3 mL/well of human ES/iPSC growth media. Cells can now be plated onto the newly coated plates. IMPORTANT: Do not allow the plates to dry out.Frequently Asked Questions (FAQs) 1. I have leftover Stem Cell Qualified ECM Gel (1:20 or 1:80 dilution). How long can I keep it?Diluted ECM Gel can be kept in the fridge for 3-4 days. Within this time frame, you can either use directly for coating or further dilute to make up the 1:80 dilution. Avoid multiple freeze thaw cycles.2. Can precoated plates be frozen for future use?No, we do not recommend freezing pre-coated plates. Pre-coated plates should be sealed with Parafilm and may be stored at 2-8°C for up to 3-4 days before use. 3. Can I use a dilution factor higher than 1:80?We recommend a maximum dilution of 1:80. Further optimization may be required for higher dilutions.4. My Stem Cell Qualified ECM Gel has already polymerized? How do I make it liquid again?Once polymerized, ECM Gel cannot revert to a liquid form again. It is recommended to start with a fresh frozen aliquot and coat the plates again. To prevent polymerization, store ECM Gel on ice during handling.5. Do I need to filter the Stem Cell Qualified ECM Gel before use?No, ECM Gel is cell culture tested and ready-to-use once diluted.，官网链接：https://www.sigmaaldrich.cn/product/mm/cc131 默克 科研、开发、生产。 作为生命科学行业的全球领先供应商，我们致力于为科研、生物技术开发和生产，以及制药药物疗法开发和生产提供各类解决方案和服务。

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