Instructions for Use:
The protocols provided below are for reference only. The actual conditions must be optimized by users based on the requirement of a particular experiment.
1.Thaw and aliquot the matrix gel
a.
Thaw the gel: Place vials of matrix gel on ice and thaw overnight at 4℃ in the dark. More time may be required to thaw matrix gel with high protein concentration. Do not place on the refrigerator door or in a refrigerator that will be opened frequently. After thawing, mix the product by vortex to ensure uniform dispersion of matrix gel.
b.
Aliquot the gel: Aliquot the uniformly dispersed matrix gel into tubes according to the amount that will be required for subsequent experiments, and store aliquot at temperatures below -20℃ in the dark. If the pipette tip is clogged or becomes inaccurate during aspiration, replace with new tips. Dispensing and subsequent manipulation of matrix gel must be performed on ice.
Note 1: The matrix gel will start to polymerize at temperature above 10℃, so it needs to be kept on ice during the experiment. Also, all pipettes, tips and consumables such as culture plates/dishes, and reagents such as culture medium or PBS used for dilution need to be pre-cooled at 4℃.
Note 2: Once the matrix gel is polymerized, immediately place on ice at 4℃ in the dark for 24-48 hours. The gelatinized matrix gel may be re-liquidated.
2.Conventional coating methods of matrix gels
There are many different coating methods for matrix gels, such as thin gel method, thick gel method, and thin coating method. Select an appropriate coating method based on the requirement of a particular experiment.
a.
Thin gel method: The gel formed by this method is about 0.5mm thick. Cells are cultured on the surface of the gel. Thin gels are mainly used for cell adhesion and proliferation, such as primary cell culture or differentiation of endothelial cells into vascular-like structures (Tube Assay), requiring a thin protein layer only.
(a)Place matrix gel on ice and thaw at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
Note: For Tube Assay, the standard matrix gel (8-13mg/ml) or high concentration matrix gel (16-26mg/ml) should be diluted to 6-8mg/ml, which can be adjusted according to actual situation.
(b)Place a culture plate/dish on ice and add matrix gel to the growth surface of plate/dish at 50μl/cm2. The volume of matrix gel required per well/dish can be referred to the following table.
|
6-well plate |
12-well plate |
24-well plate |
48-well plate |
96-well plate |
35mm dish |
60mm dish |
100mm dish |
| Well area |
~9.6cm2 |
~4.5cm2 |
~2cm2 |
~0.8cm2 |
~0.32cm2 |
~8cm2 |
~21cm2 |
~55cm2 |
| Matrix-Gel™ volume |
480μl |
225μl |
100μl |
40μl |
16μl |
400μl |
1.05ml |
2.75ml |
(c)Incubate the plate/dish at 37℃ for 30 minutes to solidify the matrix gel.
(d)(Optional) Aspirate the unbound matrix gel and rinse gently with serum-free culture medium before use.
Note: Do not scratch the surface of the gel when aspirating.
b.
Thick gel method: The gel formed by this method is about 1-2mm thick. Cells are generally cultured inside the gel, but can also be cultured on the surface of the gel. Thick gels are mainly used for 3D cell culture and Ring Assay such as differentiation of rat aortic tissue into capillary-like structures.
(a)Place matrix gel on ice and thaw at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
(b)Mix cells with matrix gel and suspend cells with prechilled pipettes.
(c)Place a culture plate/dish on ice and add the mix (Mixed Matrix-GelTM) to the growth surface of plate/dish at 150-200μl/cm2. The volume of the mix required per well/dish can be referred to the following table.
|
6-well plate |
12-well plate |
24-well plate |
48-well plate |
96-well plate |
35mm dish |
60mm dish |
100mm dish |
| Well area |
~9.6cm2 |
~4.5cm2 |
~2cm2 |
~0.8cm2 |
~0.32cm2 |
~8cm2 |
~21cm2 |
~55cm2 |
| Mixed Matrix-GelTM Volume |
1.4-2.0ml |
675-900μl |
300-400μl |
120-160μl |
48-64μl |
1.2-1.6ml |
3.1-4.2ml |
8.25-11ml |
(d)Incubate the plate/dish at 37℃ for 30 minutes to solidify the matrix gel.
(e)The culture medium can be added if necessary.
c.
Thin coating method: Use a lower concentration of matrix gel to form a mixed protein coating layer without forming a gel. Cells are cultured on this thin layer. This method can be used for cell adhesion experiments, such as the attachment and growth of human embryonic stem cells (hESC) and Transwell tumor cell invasion studies
in vitro, but may not be suitable for cell differentiation studies.
(a)Place matrix gel on ice and thaw at 4ºC. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
(b)Dilute the matrix gel to the desired concentration with serum-free culture medium or PBS.
Note: Preliminary experiments should be performed to determine the optimal concentration of matrix gel for coating.
(c)Add sufficient diluted matrix gel to the plates to ensure the entire growth surface is covered completely. Then incubate at room temperature for 1 hour to solidify the matrix gel.
(d)Aspirate the unbound matrix gel and rinse gently with serum-free culture medium before use.
Note: Do not scratch the surface of the layer when aspirating.
3.Subcutaneous tumorigenesis in immunodeficient mice (nude mice) with the matrix gel.
Subcutaneous tumorigenesis experiments are generally performed with 4-8 weeks old mice. Ethics usually require that the number of inoculated tumor cells should not exceed 1×10
7 cells per mouse. The rate of tumorigenesis varies depending on the tumorigenicity of cells, the rate of proliferation, the amount of cells inoculated, and the strain of mice. The drug intervention cycle is typically 0.5-2 months.
a.Thaw Matrix-Gel™ Basement Membrane Matrix (HC) (Beyotime,
C0382/
C0383 or
C0386/
C0387) on ice at 4ºC overnight. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
Note: High concentration matrix gels with reduced growth factors result in faster tumor formation.
b.Cell collection: Collect suspension cells directly or adherent cells after digestion with trypsin. Centrifuge the collected cell suspension at 300×g for 5 min and wash cells twice with PBS. Resuspend cells with PBS and count cell numbers. Adjust the cell density to 1×10
7-1×10
8 cells/ml. Place cells on ice until use.
Note: The collected cells should be inoculated within 1 hour.
c.Mix the cell suspension with the matrix gel at a ratio of 1:1, at which time the cell density is 0.5×10
7-5×10
7 cells/ml.
Note: The cell suspension and matrix gel mixture should be placed on ice to slow down apoptosis and prevent the matrix gel from polymerization.
d.Use an electric shaver for experimental animals (Beyotime,
FS600) to remove hair from the skin of the injection site of mice, followed by disinfection with alcohol cotton balls.
e.Use a 1ml syringe without a needle to aspirate the cell suspension and matrix gel mixture, then attach the needle.
Note: The cell suspension and matrix gel mixture should be mixed well gently by pipetting with the syringe before aspirating the mixture.
f.Perform subcutaneous injection. Inoculate100µl per mouse.
Note: The injection procedure should be as short as possible.
g.Continue to raise mice. Apparent tumor masses can be observed after 1-3 weeks. Please refer to Figure 1 for the result of subcutaneous tumorigenesis.
4.Angiogenesis in immortalized HUVEC cells with the matrix gel
a.
Starvation treatment of HUVEC cells Replace the complete culture medium with DMEM medium containing 0.2% FBS and culture cells for 24 hours.
Note: It is recommended to use the F3-F5 generation of HUVEC cells in good condition and with 70-80% fusion.
b.Place Matrix-Gel™ Basement Membrane Matrix (Standard or HC) on ice and thaw overnight at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
c.Dilute matrix gel to the desired concentration with DMEM medium.
Note 1: The best concentration of matrix gel for angiogenesis has been tested to be 6-8 mg/ml, which can be adjusted according to the actual situations.
Note 2: If Matrix-Gel™ Basement Membrane Matrix (GFR) is used for angiogenesis, appropriate growth factors can be supplemented to the culture medium to stimulate angiogenesis.
d.Place a 96-well culture plate on ice and add 50μl of diluted matrix gel per well. Incubate at 37℃ for 45-60 minutes to allow for gel polymerization.
e.Digest HUVEC cells, resuspend with DMEM medium containing 10% FBS, and count the cell number.
f.Add 50-200µl of cell suspension (30,000-50,000 cells in total) to each well.
Note 1: HUVEC cells tend to settle down easily, which can result in inconsistent cell numbers between wells. Therefore, make sure cells are suspended evenly before aspirating cells. The number of cells should not be less than 30,000 cells per well. Less cells might cause failure in forming a continuous blood vessel network.
Note 2: When dispensing the cell suspension, it is necessary to keep the pipette tip perpendicular to the top of the well and not to touch the gel.
Note 3: To improve the accuracy of the experiment, please set up at least 3 replicates.
g.Place the 96-well plate in a cell incubator and the formation of blood vessel network can be seen in 3-12 h. The time of vessel formation is closely related to the cell state. Please refer to Figure 2 for the effect of angiogenesis.
Note: The collapse of blood vessels after formation may be due to apoptosis of endothelial cells after too long period of culture.
h.At the optimal time for the formation of blood vessel network, carefully remove the culture medium, stain with DMEM culture medium containing Calcein AM (Beyotime,
C2012), and then observe under a microscope.
5.Transwell invasion assay with the matrix gel
a.
Coating transwell chambers with the matrix gel
(a)Place Matrix-Gel™ Basement Membrane Matrix (Standard) on ice and thaw overnight at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
(b)On ice, dilute the matrix gel with serum-free culture medium at a ratio of 1:8. For example, add 8µl of matrix gel to 64µl of serum-free culture medium and mix well using a prechilled pipette.
Note: Commonly used dilution factions are 1:4, 1:6, and 1:8. Other dilution factors can also be used as required by specific experiments.
(c)Add 60µl of the diluted matrix gel vertically into the Transwell chamber to ensure even spreading on the bottom of the chamber without the formation of air bubbles. Subsequently, incubate at 37℃ for 3 hours.
(d)Aspirate the unbound matrix gel and rinse gently with serum-free culture medium before use.
Note: Do not scratch the surface of the gel when aspirating.
(e)Add 100µl of serum-free culture medium and incubate the plates in a 37℃ incubator for 30 minutes for hydration.
(f)Remove the liquid from the chambers and check if any liquid has passed through the chambers into the lower chamber. If not, it is ready for cell seeding.
b.
Preparation of cell suspension
(a)(Optional) Starve cells for 12-24 hours.
(b)Digest cells and resuspend in serum-free culture medium to a final cell density of 50,000-500,000 cells/ml.
Note: Different cells have different migration abilities, and a series of cell density can be tested to determine the optimal cell density.
c.
Seeding cells
(a)Add 500µl of culture medium containing 10% FBS to the lower chamber of a 24-well plate and place the Transwell chamber inside the 24-well plate using forceps.
Note: Air bubbles are often generated during the placement of Transwell chamber, which weakens the chemotaxis of the lower culture medium significantly. Once air bubbles are generated, lift the chamber to remove air bubbles and reposition it.
(b)Add 200µl of cell suspension to Transwell chamber.
(c)Place the 24-well plate in a cell incubator for 24-48 hours.
Note: One to two hours after cell seeding, check the plate to ensure no large air bubbles are generated.
d.
Cell Fixation and staining
(a)Take out the Transwell chamber, remove the culture medium, and gently wipe the matrix gel and cells with a cotton swab.
(b)Add 600µl of 4% Paraformaldehyde (Beyotime,
P0099) to a clean well of a 24-well plate, place the chamber inside, and incubate for 20-30 minutes.
(c)Discard the fixative and wash the chamber once with PBS.
Note: Do not touch the bottom of the chamber.
(d)Add an appropriate amount of Crystalline Violet Staining Solution (Beyotime,
C0121) to a clean well of a 24-well plate, place the chamber inside, and stain for 5-10 minutes.
(e)Take out the chamber and wash it thrice with PBS.
Note: Do not touch the bottom of the chamber.
(f)Air-dry properly, and then examine by microscope. Please refer to Figure 3 for the staining result of Transwell invasion assay.
6.Organoid culture with the matrix gel
a.Place Matrix-Gel™ Basement Membrane Matrix (for Organoids) on ice and thaw overnight at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
b.Prewarm a 24-well plate in the cell incubator.
c.Take tissue (animal tissue, tumor tissue) and prepare single cell suspension of 5,000-15,000 cells per well according to the processing method of primary cells. Add an appropriate proportion of matrix gel and mix well. The ratio between matrix gel and single cell suspension can be as follow:
| Dilution ratio |
Matrix-Gel™ (For Organoid Culture) |
Single cell Suspension |
| 2:1 |
100µl |
50µl |
| 1:1 |
75µl |
75µl |
| 1:2 |
50µl |
100µl |
d.Take out the prewarmed 24-well plate from the cell incubator and immediately add an appropriate amount of the mixture dropwise to each well. Immediately incubate in a 37℃ incubator for 10-30 minutes to allow for gel polymerization.
e.After polymerization, add 500µl of culture medium suitable for the organoid per well. Incubate in the cell incubator and replace the culture medium with new ones every certain time. Please refer to Figures 4 and 5 for the results of organoid culture with the matrix gel.
7.Drug sensitivity assays of tumor organoids
a.Resuspend tumor organoids (experimental group) and normal organoids (control group) in prechilled basal culture medium at 4℃ by pipetting gently to promote gel dissolution but maintain the structural integrity of organoids. Single cell suspension can be obtained by digesting organoids with BeyoTryp™ Express Enzyme (Beyotime,
C0191/
C0192).
b.Refer to 6b-e for organoid culture. Culture plates can be 96-well plates or 384-well plates.
c.After organoid formation (in case of pipetting, the organoid will be formed after 24 hours of passaging; in case of enzymatic digestion, the organoid will be formed after 3-5 days), add different types of antitumor drugs at different concentrations to each well separately.
d.Determine the sensitivity of tumor organoids to various drugs by analyzing organoid viability with CellTiter-Lumi™ Luminescence 3D Cell Viability Assay Kit (Beyotime,
C0061/
C0062) or by live cell imaging with fluorescent dye and High-content screening (HCS).
8.3D cell culture with matrix gel
Note: This protocol is provided for culturing cells in 96-well plates. For other types of culture vessels, adjustments can be made accordingly, or refer to "2b. Thick Gel Method" directly.
a.Cultivate cells under normal culture conditions according to the characteristics of the cells.
b.Digest and collect cells, resuspend cells with serum-free culture medium, count the cell number, and aspirate the cell suspension as needed for the experiment. Centrifuge at 300×g for 5 min, discard the supernatant, and place on ice.
c.Place Matrix-Gel™ Basement Membrane Matrix (Standard or High Concentration) on ice and thaw overnight at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
d.Mix the matrix gel with the serum-free culture medium at a ratio of 1:1, and mix well.
Note: The commonly used dilution ratio is 1:1 and 1:2. It can be adjusted as required by the experiment.
e.Gently resuspend the cell pellet with an appropriate amount of diluted matrix gel, and avoid the formation of air bubbles. The recommended cell density after resuspension is 5000 cells per 10μl of resuspension. Place cells on ice after resuspension. The resuspension time should not exceed 30 seconds to avoid gel solidification.
Note: The cell density can be adjusted as required by the experiment.
f.Place a 96-well plate on ice and add 8 μl of cell suspension dropwise and vertically to the center of each well, then spread slowly and evenly with a pipette tip. Carefully invert the 96-well plate and incubate at 37℃ for 30 minutes to allow for gel polymerization.
g.After polymerization, add 60μl of prewarmed culture medium containing 10% FBS per well and supplement the marginal empty wells with sterile water.
Note: Compounds to be tested can be added at this step.
h.Incubate the culture plate in a 37℃ incubator and examine the formation of 3D structure every day.
i.Replace with new culture medium every 4 days (day 4, 8, 12).
j.Analyze cells when the cells grow to an ideal stage. Please refer to Figure 6 for the 3D tumor sphere formed with the matix gel.
9.Culture of induced Pluripotency Stem Cell (iPSC) with the matrix gel
Note 1: We do not recommend using antibiotics for iPSC culture, as antibiotics can affect the activity and the differentiation potential of cells.
Note 2: The iPSC should be cultured in an isolated environment from other cells and assayed for mycoplasma after two passages.
a.Coating multi-well plates with the matrix gel
(a)Place Matrix-Gel™ Basement Membrane Matrix (For Stem Cells) on ice and thaw overnight at 4℃. Make matrix gel homogeneous by pipetting with prechilled pipettes or tips.
(b)On ice, mix the matrix gel with DMEM culture medium at a ratio of 1:80 or 1:100 (e.g., take 1 ml of matrix gel and add it to 80 ml or 100 ml of DMEM culture medium), and use a pre-chilled pipette to mix until it is homogeneous.
Note: The dilution ratio can be adjusted as required by the experiment.
(c)Place a 6-well plate on ice and add 1 ml of diluted matrix gel per well.
(d)Incubate at 37℃ overnight.
Note: Normally, incubation for 1 hour is sufficient, but overnight incubation gives better results for cell culture.
(e)Aspirate the unbound matrix gel and rinse gently with serum-free culture medium before use.
Note: Do not scratch the surface of the gel when aspirating.
b.
Preparation of Y-27632 (ROCK inhibitor) solution: Prepare a 10mM Y-27632 solution by dissolving Y-27632 (
SC0326) in PBS. For example, dissolve 2.47mg of Y-27632 in 1ml of PBS to obtain 1ml of 10mM Y-27632 solution.
Note: The working concentration of Y-27632 is 10µM.
c.
Preparation of human embryonic stem cell (hECS) culture medium (e.g., mTeSR™1 or TeSR™-E8™) containing Y-27632: Mix Y-27632 solution (10mM) with the mTeSR™1 culture medium at a ratio of 1:1000. For example, add 10µl of Y-27632 solution into 10ml of mTeSR™1 culture medium and mix well.
d.
iPSC recovery
(a)Thaw the frozen iPSC in a 37℃ water bath, transfer all the content to a centrifuge tube, and centrifuge at 300×g for 5 min at room temperature.
(b)Discard supernatant and resuspend iPSC with 2 ml of mTeSR™1 culture medium containing Y-27632. Subsequently, transfer cell suspension to one well of a 6-well plate coated with matrix gel and incubate in a cell incubator.
Note: It is recommended to transfer one tube of cells (approximately 1 million) to one well of a 6-well plate to maximize cell viability.
(c)Replace the Y-27632-containing mTeSR™1 culture medium with Y-27632-free mTeSR™1 culture medium on the next day. Please refer to Figure 7 for the culture effect of ES cells.
e.
iPSC passaging
Note: iPSC cells will rapidly differentiate and die after growing to monolayer. Therefore, cells need to be passaged before that to maintain their activity and pluripotency.
(a)Remove supernatant, wash cells once with 1 ml of PBS, and add 1 ml of appropriate cell digestion buffer such as BeyoTryp™ Express Enzyme (Beyotime,
C0191/
C0192) or Accutase.
(b)Transfer the culture plate to a cell incubator and incubate for 3 minutes. Observe under the microscope, and most of the cells should be dissociated from the surface of wells. If the cells are still attached, gently tap the bottom of the culture plate to dissociate cells.
(c)Transfer cells to a centrifuge tube, wash the surface of the culture plate twice with DMEM culture medium and transfer to the same centrifuge tube. Centrifuge at 300×g for 5 min at room temperature.
(d)Discard the supernatant and resuspend with mTeSR™1 culture medium containing Y-27632. Subsequently, transfer the cell suspension to a 6-well plate coated with matrix gel and incubate in a cell incubator.
f.
iPSC cryopreservation
(a)Preparation of cell freezing medium: 20% FBS + 10% DMSO + 70% DMEM or 90% FBS + 10% DMSO. Beyotime’s Cell Freezing Medium (
C0210) is recommended.
(b)Digest cells according to the instructions of cell digestion buffer.
(c)Count cells, discard supernatant, and add an appropriate amount of cell freezing medium to resuspend cells. Dispense cell resuspension into cryogenic vials at 1 ml per tube (approximately 1 million cells).
(d)Place the cryogenic vials in a device that allows for gradual temperature reduction at approximately 1℃/min in a -80℃ refrigerator. BeyoCool™ Cell Freezing Container (
FCFC012) is recommended.
(e)Place in a -80℃ refrigerator for approximately 24 hours and then transfer to liquid nitrogen for storage.
FAQ：
I.Basic questions about the usage of matrix gel
1.Can Matrix-Gel™ Basement Membrane Matrix be stored at -70℃?
Yes, Matrix-Gel™ Basement Membrane Matrix can be stored at -70º C. We recommend aliquoting the maxtrix gel and store them in polypropylene (PP) or other ultra-low temperature tolerant microfuge tubes.
2.Can Matrix-Gel™ Basement Membrane Matrix be repeatedly frozen and thawed?
Repeated freeze-thaw should be avoided as much as possible. We recommend aliquoting the matrix gel after the first thawing.
3.How should I store unused Matrix-Gel™ Basement Membrane Matrix?
It is not recommended to keep matrix gels that have been mixed with culture medium or buffer for future use.
4.How should I pipette Matrix-Gel™ gels?
We recommend using prechilled syringes, pipettes or tips for pipetting. Do not touch the bottom of the bottle when aspirating and do not dispense the liquid too quickly or with too much force.
5.Do I need to prechill consumables such as culture plates/dishes, tips and centrifuge tubes when using Matrix-Gel™ matrix gels?
Yes. Because Matrix-Gel™ matrix gels will begin to polymerize at temperatures above 10℃. It is recommended to use prechilled pipettes, tips and centrifuge tubes when handling.
6.Can Matrix-Gel™ matrix gels polymerize quickly?
Matrix-Gel™ matrix gels polymerize rapidly at 22-35℃.
7.How to dilute Matrix-Gel™ matrix gels?
Dilute Matrix-Gel™ matrix gel with serum-free culture medium or PBS prechilled on ice.
8.How long can the matrix gel-coated plates/dishes be stored?
The coated plates/dishes are best to be used on the same day as they are prepared. If temporarily not used, store in 37℃ incubator for a maximum of 7 days. For storage, the surface of the gel needs to be evenly covered with serum-free culture medium to keep it moist.
9.What is the minimum concentration for Matrix-Gel™ to polymerize?
The minimum gel-forming concentration of Matrix-Gel™ is 3 mg/ml. Different gel concentrations are required for different experimental purposes. Matrix-Gel™ concentration varies from batch to batch and the amount of dilution buffer (e.g., PBS or serum-free medium) to be added should be calculated based on the final working concentration (mg/ml). For Matrix-Gel™ matrix gels used in
in vivo studies, the final working concentration should not be less than 4 mg/ml to ensure complete gel polymerization.
10.How long can Matrix-Gel™ matrix gels be maintained in the body?
At least 1 week.
11.Why does Matrix-Gel™ Basement Membrane Matrix form gel at 37℃, but is liquid at 4℃?
Matrix-Gel™ is a basement membrane matrix derived from mouse EHS tumors, comprising laminin, type IV collagen, nestin, heparan sulfate proteoglycans, epidermal growth factor, insulin-like growth factor, and other growth factors. These proteins form the basic structure of the matrix gel. At 22-37℃, the macromolecules can be chemically bonded to each other, leading to gel formation of matrix gels. At low temperatures (e.g. 4℃), the matrix gel appears liquid because there is not enough energy to induce chemical bonding.
12.Why is Matrix-Gel™ matrix gel viscous?
The higher the protein concentration of Matrix-Gel, the more viscous the gel. Matrix-Gel™ products are viscous unless diluted. High concentrations of Matrix-Gel™ can be used for cell culture or
in vivo injection without dilution, or can be diluted to any protein concentration range and used as a standard Matrix-Gel™ product. The optimal dilution factor of Matrix-Gel should be determined by users based on the experimental needs.
In addition to being viscous due to high protein concentrations, the condition of the matrix gel is also related to temperature changes during transport and storage conditions. It must be shipped with dry ice. If the refrigerator in which Matrix-Gel™ Matrix Gel is stored has an automatic defrost function, the temperature of the refrigerator may rise during defrosting and may cause the Matrix-Gel™ to polymerize. Therefore, do not store Matrix-Gel™ matrix gels in such refrigerators. To ensure the effectiveness of Matrix-Gel™, repeated freeze-thaw should be avoided as much as possible. Upon receipt of a new Matrix-Gel™ Matrix Gel, dispense it immediately. Matrix-Gel™ should be thawed on ice.
13.Why didn't the cells adhere to the wall and the Matrix-Gel™ was detached?
First of all, it is necessary to check whether too much cells were seeded. The amount of matrix gel should be equal to the amount of culture medium used for cell culture. If the matrix gel is diluted too much, the gel formed will easily detach from the surface of the culture vessel.
14.How to deal with the precipitation that appears in undiluted Matrix-Gel™ Basement Membrane Matrix?
Centrifuge at low speed at 4℃ to remove the precipitate.
15.What is the refractive index of Matrix-Gel™ ?
At 20℃, Matrix-Gel™ has a refractive index of approximately 1.3406-1.3407 and a relative refractive index of approximately 1.0056 (under equivalent conditions, water has an index of 1.333).
16.Does Matrix-Gel™ have autofluorescence?
Matrix-Gel™ is a protein mixture that has been dialyzed and dissolved in DMEM culture medium. To prevent microbial contamination, the culture medium is supplemented with gentamicin. Therefore, the components of Matrix-Gel that may trigger fluorescence are protein, vitamin, and gentamicin (an aminoglycoside antibiotic). If fluorescence needs to be used to detect cell growth status, it is recommended to set up a control to exclude background fluorescence.
II.Questions about the application of matrix gel
1.When do I need to use phenol red-free Matrix-Gel™?
Phenol Red-free Matrix-Gel™ is recommended for experiments involving color detection, such as the quantification of endothelial tubulogenesis using fluorescent dyes and the phenol red-free Matrix-Gel™ must be used for endometrial cell culture. Phenol red is structurally similar to some non-steroidal estrogens, has significant estrogenic activity, and may have the ability to interfere with endocrine and hormonal metabolism in experimental animals, so phenol red-free Matrix-Gel™ can also be used for some
in vivo applications. In addition, phenol red is a potential endocrine disruptor that may interfere with the natural production and metabolism of hormones in experimental animals.
2.Which experiments can high concentration Matrix-Gel™ be used for?
High concentration Matrix-Gel™ can be used for
in vivo applications such as 3D cell culture and
in vivo tumorigenesis in nude mice. The high concentration Matrix-Gel™ maintains its integrity and remains in situ after subcutaneous injection into mice along with tumor cells to facilitate in situ analysis or future tumor sampling.
3.How to select the thin/thick gel method and what are the applications of 3D cell culture?
The thin gel method is mainly used for cell adhesion and proliferation, e.g., primary cell culture requires only a thin protein layer to assist rather than a protein matrix. The thick gel method is mainly used for 3D cell culture and ring assay, such as the differentiation of rat aortic tissue into capillary-like structures and cell invasion, etc. 3D cell culture experiments are mainly used to study cell-cell interactions and complex structures, etc.
4.Can Matrix-Gel™ be used in studies of ES/iPS cell differentiation induction?
Yes, we recommend using Beyotime’s Matrix-Gel™ Basement Membrane Matrix (For Stem Cells) (
C0391/
C0392).
5.How to harvest cells from Matrix-Gel™ ?
Neutral proteases such as Beyotime’s Dispersease (
ST2339) or BeyoTryp™ Express Enzyme (
C0191/
C0192) are recommended for harvesting cells cultured in Matrix-Gel™. Neutral protease and BeyoTryp™ Express Enzyme are able to obtain single cell suspensions more gently and efficiently than trypsin, collagenase or other protein hydrolases, without damaging cells or splitting cell surface proteins. Neutral protease or BeyoTryp™ Express Enzyme is not damaging to cells that need to be further inoculated or bioassay. Additionally, neutral protease or BeyoTryp™ Express Enzyme can be used for tissue isolation. Metabolic assays and RNA recovery experiments can also be performed using the appropriate cell recovery solution. It is recommended to use the cell recovery solution at 4℃ for non-enzymatic cell harvesting.
Other methods: Depolymerize the Matrix-Gel™ at 4-6℃, which takes time and is only suitable for some applications, or centrifuge to disrupt the matrix gel structure.
6.How to fix the cells cultured with Matrix-Gel™ for immunohistochemistry and immunofluorescence assays? How to avoid depolymerization?
Fixation can be performed using 2% paraformaldehyde (e.g., fix with 2% paraformaldehyde prepared in PBS at room temperature for 20 minutes). In some cases, depolymerization may occur after fixation and 1% glutaraldehyde can be added (Glutaraldehyde is commonly used as a fixative for electron microscopy). If immunofluorescence assay is required, the addition of glutaraldehyde will result in significant background fluorescence. To solve this problem, we recommend using NaBH4 for quenching after fixation. NaBH4 is extremely bubbly and this step must be performed with care on a horizontal table to avoid shaking and minimize bubble formation. Alternatively, a lower concentration (e.g., 0.1% to 0.5%) of glutaraldehyde can be attempted. The lower the concentration, the lower the background fluorescence signal.
7.Will the extraction process cause denaturation of laminin?
No, laminin will not be denatured during the extraction process.
III.Questions about the composition of the matrix gel
1.Does Matrix-Gel™ contain DNA or RNA?
Yes, Matrix-Gel™ has not been digested with DNase or RNase and may contain traces of DNA and RNA. Thus, a Matrix-Gel™ control group without cells should be prepared when performing RNA analysis.
2.Does Matrix-Gel™ matrix gel contain vascular endothelial growth factor (VEGF) and metalloproteinases (MMPs)?
The standard Matrix-Gel™ contains 5.0-7.5 ng/ml of VEGF and the low growth factor Matrix-Gel™ contains 1.0-1.5 ng/ml of VEGF. The Matrix-Gel™ may also contain traces of mouse tumor cell-derived metalloproteinases (MMPs).
3.Does Matrix-Gel™ contain LDEV?
No. It has been confirmed by Immunoassay and PCR that Matrix-Gel™ does not contain lactate dehydrogenase elevated virus (LDEV) or lactate dehydrogenase elevated virus (LDHV). No bacteria, fungi or mycoplasma have been detected.
4.Is there any urea in Matrix-Gel™?
No.
5.What buffer is used in Matrix-Gel™?
Low sugar DMEM (1g/L) which contains 50µg/ml gentamicin.
6.Does Matrix-Gel™ contain Fibronectin?
Yes, trace amounts of Fibronectin have been detected in Matrix-Gel™ by Western Blot.
7.Does Matrix-Gel™ contain vitreous binding protein (Vitronectin)?
Some tissues may contain traces of blood and therefore traces of Vitronectin may be present in Matrix-Gel™.
8.What other substances are present in Matrix-Gel™ matrix gels?
Matrix-Gel™ may also contain trichloromethane at concentrations less than 0.02%, as well as other unknown proteins or molecules produced by tumor cells.
References：
1.Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. Adv Drug Deliv Rev. 2014. 79-80:3-18.
2.Hughes CS, Postovit LM, Lajoie GA. Proteomics. 2010. 10(9):1886-1890.
3.Biederer T, Scheiffele P. Nat Protoc. 2007. 2(3):670-6.
4.Yu X, Sidhu JS, Hong S, Faustman EM. Toxicol Sci. 2005. 84(2):378-93.
5.Roskelley CD, Desprez PY, Bissell MJ. Proc Natl Acad Sci U S A. 1994. 91(26):12378-82.
6.Debnath J, Muthuswamy SK, Brugge JS. Methods. 2003. 30(3):256-68.
7.Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, et al. Biochemistry. 1982. 21(24):6188-6193.
8.Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, et al. Biochemistry. 1986. 25(2):312-8.
9.Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, et al. Exp Cell Res. 1992. 202(1):1-8.
10.McGuire PG, Seeds NW. J Cell Biochem. 1989. 40(2):215-227.
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