Product Introduction
Type
Growth Factor Reduced (GFR)
Product
ECM-Gel
ECM-Gel
Catalog
LV-6829-1-05ml
LV-6829-1-10ml
Specification
5mL
10 mL
Concentration of protein
8-10mg/mL
8-10mg/mL
Free shipping or not
Over 3000 free shipping
Over 3000 free shipping
Delivery
1 week
1 week
Carriage
Transportation of Dry ice
Transportation of Dry ice
Storage
-70 ℃ to -20 ℃
-70 ℃ to -20 ℃
Life cycle
24 months
24 months
Warranty
21 months
21 months
ECM-Gel
Store at -20℃. Avoid repeated freezing and thawing. For Research Use Only.
I Introduction
ECM-Gel is a soluble substrate membrane preparation extracted from healthy pigs, which shapes into colloidal forming above 10℃. Compared to rodents, pigs are closer to humans in terms of genetic distance. ECM-Gel is naturally substitutable and has a wide-spread applications in clinical transplantation, medicine, cosmetic medicine and pharmaceutical auxiliary materials. Moreover, there are hundreds of literatures have been published on Pubmed at present, indicating that the ECM-Gel of pig origin are naturally replaceable. Based on our decellularization technology, as well as standardized, large-scale production and manufacture experience, we ensure the excellent quality, uniformity, consistency and stability of products, offering the scientists another choice.
The major components of this product are laminin, type IV collagen, nestin and heparan sulfate proteoglycan, with the advantages of low endotoxin and DNA content. It is mainly used to support cell growth and differentiation in vitro, metabolic/toxicology studies, cell adhesion, angiogenesis, cells migration/invasion and in vivo tumorigenesis experiments, etc.
ECM-Gel Products and their Applications:
Type |
ECM-Gel |
Cat.no. |
Size |
Applications |
Standard |
ECM-Gel |
LV-6828-1 |
5/10ml |
Cell growth, differentiation, invasion, morphology, function, gene expression, etc. Protein concentration is 8-10mg/ml. |
ECM-Gel phenol red-free |
LV-6828-2 |
5/10ml |
||
Growth Factor Reduced (GFR) |
ECM-Gel |
LV-6829-1 |
5/10ml |
Suitable for higher requirements of ECM-Gel experiments, reduce the interference of cytokines;Protein concentration is 8-10mg/ml. |
ECM-Gel phenol red-free |
LV-6829-2 |
5/10ml |
||
Standard, High Concentration |
ECM-Gel |
LV-6830-1 |
5/10ml |
In vivo experiments, angiogenesis, culture of tumor organoids, etc. Better support for 3D cell culture, cells grow better in 3D environment;Protein concentration is 16-20mg/ml. |
ECM-Gel phenol red-free |
LV-6830-2 |
5/10ml |
||
GFR, High Concentration |
ECM-Gel |
LV-6830-3 |
5/10ml |
|
ECM-Gel phenol red-free |
LV-6830-4 |
5/10ml |
Remarks: Our ECM-Gel products are derived from healthy pigs, not mouse EHS tumor tissue. The matrix collagen composition and proportion are slightly different, and the optimal gelatinization concentration is slightly lower than the Matrix-gel concentration.
II Reagents and Materials
-Serum-free medium
-Vortex mixer
-Pre-chilled centrifugal tube
-Culture plate
-Pipette
-Pre-chilled tip
-Pipettor
-Biosafety cabinet
-CO2 incubator (37℃/5%)
III Methods
Precautions:
1) Adequate melting of the ECM-Gel requires at least 4℃ for overnight. In some cases, the product concentration is high, it takes longer to melt.
2) Throughout the operation, all culture vessels, consumables and mediums in contact with the ECM-Gel should be pre-cooled, as the ECM-Gel will begin to gel at more than 10℃.
3) The optimal concentration of matrix gelatinization in vitro is 6 mg/ml, and the in vivo gelatinization should not be less than 3 mg/ml.
4) For your safety and health, please wear a lab coat, a disposable mask and gloves, and take necessary precautions.
Thawing
Transfer the ECM-Gel product from the -20 °C refrigerator to the 4 °C refrigerator (on ice) for overnight thawing (it may take more time when the protein concentration is high), and shake the vial with vortex after liquefaction to ensure that the ECM-Gel is evenly dispersed. If you can't use it all at a time, you can divide it on ice once and freeze-thaw it only once. If the ECM-Gel has more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes. Place the ECM-Gel in a sterile area and set it aside on ice.
Caution: Be careful not to over-dry the ECM-Gel during the gelling stage. The melted ECM-Gel will gradually solidify above 10 °C. And the ECM-Gel should always be placed on ice throughout the experimental process.
Gelling
1. Thin layer gel method (thickness 0.5 mm)
1)Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes.
2)Place the culture plate on ice, and add ECM-Gel to the culture plate according to 50μl of ECM-Gel/cm2 to ensure that the colloid is evenly coated at the bottom of the culture well.
3)Place the culture plate in a 37℃ incubator for 30-40 minutes to make the ECM-Gel completely gelatinous.
4)Gently wash the culture wells with serum-free medium to remove the unsolidified matrix proteins. Make sure that the tip of the pipette does not scratch the glue. The plates are now ready to use.
2. Thin layer coating method (standard/growth factor reduced 8-10mg/ml)
1)Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes.
2)Transwell coating:
Dilute the ECM-Gel with pre-chilled serum-free medium in the ratio of 1:6-1:8 (protein coating amount of 100-400μg/cm2). Then, add the diluted ECM-Gel to the Transwell chamber, taking the 24-well plate as an example, 50-100 μl can be added to each chamber.. After that, place it in a 37℃ incubator for 30-40 minutes to make the protein to be fully coated.
3)The coated Transwell or culture plate should be hydrated with serum-free culture in a 37℃ incubator for 30 minutes before use , followed by subsequent migration invasion experiments.
4)Digest treated cells (can be starved for 12-24 h first), make a single cell suspension using serum-free medium containing 0.05%-0.2% BSA, and seed into a transwell chamber in the appropriate number of cells per well.
5)Add 600 μL of 10% FBS medium to the lower chamber of the 24-well plate, and avoid the formation of bubbles between the lower culture medium and the small chamber when adding, and the production of air bubbles is easy to weaken or even disappear the chemotactic effect of the lower culture medium.
6)Cultured cells: 12-48h in normal culture (time depends mainly on the ability of cancer cells to invade).
7)Remove the transwell chamber, discard the culture medium in the wells, wash the PBS 2 times, fix the 4% paraformaldehyde for 30 min, and air dry the chamber appropriately; 0.1% crystal violet staining for 15 min, PBS rinsed, gently wiped off the upper layer of unregranted cells with a cotton swab, and randomly selected 5 field statistics under the microscope.
8):The protein coating amount is from 50μg/cm2 to 500μg/cm2 (cm2 is the culture base area). The optimal amount of coating needs to be optimized according to the experience of the researcher or the individual needs of the researcher.
3. Thick layer gel method (1.0 mm, mainly used for 3D cell, organoid culture)
1)Use pre-cooled tip or pipette and gently aspirate the ECM-Gel to mix evenly (whenever the ECM-Gel blocks the tip or pipette, please change the tip or pipette). If the ECM-Gel has more bubbles, they can be removed by centrifugation at a low temperature of 400g for 5 minutes.
2)Place the culture plate on ice and use a pre-chilled tip to add ECM-Gel to the pre-chilled cell suspension (ECM-Gel: the cell suspension volume is 4:1-2:1, the optimal volume ratio is 3:1). Then, gently mix well.
3)Add cell-containing ECM-Gel to the culture plate at 150-200μl/cm2 (cm2 is the culture floor area). Place the plate in a 37℃ incubator for 30-40 minutes to gel.