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形状
gel form
テクニック
cell culture | stem cell: suitable
詳細
Stem Cell Qualified ECM Gel Coating Protocol
NOTE: 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 mL
NOTE: 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.
NOTE: 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 mL
NOTE: 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.
Stem Cell Qualified ECM Gel is a soluble basement membrane extract (BME) purified from Engelbreth-Holm-Swarm (EHS) tumor that has been pre-qualified for human ES/iPS cell culture. The matrix polymerizes at 20-40°C to form a reconstituted basement membrane. ECM Gel contains laminin, collagen IV, entactin and heparan sulfate among other ECMs. The matrix has been extensively tested and optimized for human ES/iPS cell culture.
Features and Benefits
• Eliminates the need for time-consuming prescreening and lot identification.
• Provides an optimized ECM coating necessary for long term culture of pluripotent human ES/iPS cells.
• Supports feeder-free culture of human ES/iPS cells in serum-free media.
• Compatible with all feeder free human ES/iPS cell expansion media (i.e. mTeSR®, PluriSTEM)
Features and Benefits
• Eliminates the need for time-consuming prescreening and lot identification.
• Provides an optimized ECM coating necessary for long term culture of pluripotent human ES/iPS cells.
• Supports feeder-free culture of human ES/iPS cells in serum-free media.
• Compatible with all feeder free human ES/iPS cell expansion media (i.e. mTeSR®, PluriSTEM)
アプリケーション
Research Category
幹細胞研究
幹細胞研究
品質
Appearance: Red, clear of particulates
Endotoxin Level: <20 EU/mL
Microbial Contamination: Pass
Mycoplasma: Negative
Functional Assay: <5% spontaneous differentiation for 3 passages using Human iPS cells
Endotoxin Level: <20 EU/mL
Microbial Contamination: Pass
Mycoplasma: Negative
Functional Assay: <5% spontaneous differentiation for 3 passages using Human iPS cells
保管および安定性
Store at -20ºC. Thaw overnight on ice in a 2-8°C refrigerator. Minimize freeze thaws by aliquoting into one-time use aliquots. Store aliquots in either a -20°C or -80°C freezer until ready for use. Do not store in a frost-free freezer.
Note: Stem Cell Qualified ECM Gel is extremely temeprature sensitive and may polymerize at room temperature or higher temperature within 5-10 minutes. Keep on ice at all times and use prechilled pipette tips, tubes and plates.
Note: Stem Cell Qualified ECM Gel is extremely temeprature sensitive and may polymerize at room temperature or higher temperature within 5-10 minutes. Keep on ice at all times and use prechilled pipette tips, tubes and plates.
法的情報
mTeSR is a registered trademark of WiCell Research Institute, Inc.
免責事項
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
保管分類コード
12 - Non Combustible Liquids
WGK
WGK 1
引火点(°F)
Not applicable
引火点(℃)
Not applicable
適用法令
試験研究用途を考慮した関連法令を主に挙げております。化学物質以外については、一部の情報のみ提供しています。 製品を安全かつ合法的に使用することは、使用者の義務です。最新情報により修正される場合があります。WEBの反映には時間を要することがあるため、適宜SDSをご参照ください。
Jan Code
CC131-5ML-10P:
CC131-5ML:
試験成績書（COA）
製品のロット番号・バッチ番号を入力して、試験成績書（COA） を検索できます。ロット番号・バッチ番号は、製品ラベルに「Lot」または「Batch」に続いて記載されています。
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Hynda K Kleinman et al.
Seminars in cancer biology, 15(5), 378-386 (2005-06-25)
The basement membrane extracellular matrix contacts epithelial, endothelial, fat and smooth muscle cells. Because this extracellular matrix is so thin, it had been hard to study its composition, structure, and function. An extract of a tumor was found to contain
資料
Cell culture protocols using Sigma ECM Gel Matrix EHS basement membrane extract (BME) for neurite outgrowth, cell invasion and angiogenesis tube formation assays.
Skip weekend feedings. Defined serum-free and feeder-free expansion media for human pluripotent stem cells (ES and iPS cells). See publications and protocols.
Skip weekend feedings. Defined serum-free and feeder-free expansion media for human pluripotent stem cells (ES and iPS cells). See publications and protocols.
ライフサイエンス、有機合成、材料科学、クロマトグラフィー、分析など、あらゆる分野の研究に経験のあるメンバーがおります。.
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