CN113913361A - Biological kit for researching cell mechanical force induction and preparation method thereof - Google Patents
Biological kit for researching cell mechanical force induction and preparation method thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention discloses a biological kit for researching cell mechanical force induction and a preparation method thereof, wherein the biological kit comprises a biological glue matrix, and the biological glue matrix comprises the following components of 3-10 wt% of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide; 30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate; 2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water. The invention adopts a plurality of chemical substances to combine to generate hydrogel with different hardness degrees, constructs a biological glue matrix which is nontoxic, transparent and can be adhered to glass and can be used for cell growth, and can serve the cell experimental research in the field of biological life science. The biological glue matrix can adjust the proportion according to the experimental requirements, and the kit has the advantages of simplicity, repeatability, stable effect, no biological cytotoxicity, strong adhesion, capability of staining cells and the like.
Description
Technical Field
The invention relates to the technical field of cell biological kits, in particular to a biological kit for researching cell mechanical force induction and a preparation method thereof.
Background
More and more studies have shown that Mechanical stress (Ms) plays an important role in physiological processes such as cell proliferation, differentiation, apoptosis, gene expression, and tissue growth, functional integration, and in certain pathophysiological processes such as myocardial hypertrophy, atherosclerosis and skin expansion, and wound repair of bone fracture. However, the exact response mechanism of how the stress is sensed and transmitted into the cell, which ultimately leads to a series of biological effects in the cell, is still under investigation and is not fully elucidated.
Human cells grow in a micro-dynamic environment provided by an organism, and mechanical stress can not only cause the change of cell morphology and structure, but also regulate and control the functional state of the cells, influence the proliferation, differentiation and apoptosis of the cells and play an important role in certain physiological and pathological processes.
The biological basis and possible mechanism of cellular mechanical stress response, which indicates the tensile integrity of cellular structure, integrin, secondary messenger system, stress response element, etc. are the biological basis of cellular mechanical stress response, including cell membrane ion channel (especially the tension activated cation selective channel, SA-cat), phospholipase C channel coupled with G protein, intracellular calcium ion and cytoskeleton, etc. all participate in the mechanical signal transduction of cells, and the extracellular matrix-integrin-cytoskeleton complex is the main mechanical signal transduction pathway.
The mechanical force sensing of cells on different growth matrixes has important biological significance on the self regulation of the cells, for example, the mechanical force sensing channels on the surfaces of the cells are Trpv4, Stom3, Stom1, Kcnk4 and other channels sense the soft and hard matrixes for the survival of the cells in vivo. However, an effective and simple in-vitro simulation environment for how mechanical force regulates the self-biological process of cells is still lacked, and a good biological kit research tool is still lacked in the field.
Disclosure of Invention
The invention aims to provide a biological kit for researching the cell mechanical force induction.
To achieve the above objects, in one embodiment, the present invention provides a biological kit for studying cellular mechanical force induction, comprising a biogel matrix, wherein the biogel matrix comprises the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
In another embodiment of the invention, a biological kit for studying the mechanical force response of cells is used, comprising:
a glass sheet;
the coating liquid box is used for storing the first coating liquid and the second coating liquid;
a biogel substrate box for storing reagents for preparing the biogel substrate;
a perforated plate;
the lavage liquid box is used for storing a first lavage liquid and a second lavage liquid;
the activation liquid box is used for storing a first activation liquid and a second activation liquid;
the matrix incubation liquid is used for storing the matrix incubation liquid, and comprises a matrix coating liquid and a Laminin reagent;
the biological glue matrix comprises the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
In a preferred embodiment of the present invention, the biogel matrix comprises the following components:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
Based on the biological kit, the invention also discloses a preparation method of the biological kit for researching the cell mechanical force induction, which comprises the following steps:
immersing the glass sheet in the first coating liquid for treatment for several minutes, and then taking out and airing; then immersing the glass sheet in a second coating solution for processing for a plurality of minutes, taking out and airing to obtain a coated glass sheet;
step (2), preparing a biological glue matrix;
uniformly dripping the biological glue matrix on a coated glass sheet, covering the uncoated glass sheet on the biological glue matrix, flattening the biological glue matrix, and naturally airing until the biological glue matrix is solidified; uncovering the uncoated glass sheet after solidification, and adhering the biological adhesive matrix on the coated glass sheet;
step (4), placing the coated glass sheet adhered with the biological glue matrix in the holes of the porous plate, and performing first lavage by using a first lavage liquid;
pouring the first lavage fluid, then dripping the first activation fluid for first activation, pouring the first activation fluid after the first activation, and then dripping the second activation fluid;
pouring out the second activating solution, and dropwise adding a matrix incubation solution for incubation; pouring out the matrix incubation liquid after incubation is finished, then using the second lavage liquid for carrying out second lavage, and pouring out the second lavage liquid after lavage is finished; and then packaging.
In the preferable scheme of the invention, the immersion time of the glass sheet in the first coating liquid in the step (1) is 5-20 min; the immersion time of the glass sheet in the second coating liquid is 5 min-20 min;
the first coating solution is 70% ethanol solution, and contains 0.1 mol/L-0.3 mol/L NaOH;
the second coating solution is 95% ethanol solution, and contains 0.5-1.5% wt of affinity silane and 3-6% wt of glacial acetic acid.
In a preferable scheme of the invention, the immersion time of the glass sheet in the first coating liquid in the step (1) is 10 min; the immersion time of the glass sheet in the second coating liquid is 10 min;
the first coating solution is 70% ethanol solution, and contains 0.2ol/L NaOH;
the second coating solution is a 95% ethanol solution, and contains 1.2% wt of affinity silane and 5% wt of glacial acetic acid.
In a preferred embodiment of the present invention, the biogel matrix is prepared from the following raw materials:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water;
the raw materials of the biological glue matrix are added in sequence and then mixed evenly to obtain the biological glue matrix.
In a preferred embodiment of the present invention, the biogel matrix is prepared from the following raw materials:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
In a preferred scheme of the invention, the first lavage fluid is methanol;
the second lavage liquid is serum-free DMEM solution;
the solvent of the first activating solution is double distilled water containing 10mmol/L morpholine ethanesulfonic acid and 500mmol/L NaCl;
the solvent of the second activating solution is double distilled water containing morpholine ethanesulfonic acid 10mmol/L, Nacl500mmol/L, N-hydroxysuccinimide 0.48 mol/L; n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride 0.2 mol/L.
The matrix incubation liquid comprises matrix coating liquid and a Laminin reagent; the substrate coating solution is HEPES buffer solution with the concentration of 50mmol/L, and 0.1mol/L glycine is added into the HEPES buffer solution; the concentration of the Laminin reagent dissolved in the matrix coating liquid is 50 ug/ml;
in the preferable scheme of the invention, the first lavage time is 5 min-15 min;
the second lavage lasts for 4-6 h;
the treatment time of the first activating solution is 5-15 min;
the treatment time of the second activating solution is 5-15 min;
the incubation time of the substrate incubation liquid is 12-18 h.
In summary, the invention has the following advantages:
1. the invention adopts a plurality of chemical substances to combine to generate hydrogel with different hardness degrees, constructs a biological glue matrix which is nontoxic, transparent and can be adhered to glass and can be used for cell growth, and can serve the cell experimental research in the field of biological life science.
2. The biological glue matrix can adjust the proportion according to the experimental requirements, and the kit has the advantages of simplicity, repeatability, stable effect, no biological cytotoxicity, strong adhesion, capability of staining cells and the like.
Drawings
FIG. 1 is an enlarged, live-action view of a biogel matrix according to an embodiment of the present invention, the scale in the live-action view being 2 mm;
FIG. 2a is a schematic representation of the growth of a cytoplasmic matrix on a softer matrix of biogel according to the invention in Experimental example 1; FIG. 2b is a schematic representation of cytoplasmic matrix growth on a hard biogel matrix;
FIG. 3 shows the results of the test in Experimental example 2 of the present invention;
FIG. 4 is an enlarged view of a portion of FIG. 3 in accordance with the present invention;
FIG. 5 shows the results of immunofluorescent staining assay in Experimental example 3 of the present invention;
FIG. 6 shows the results of real-time calcium imaging in Experimental example 3;
FIG. 7 shows the results of the test in Experimental example 4 of the present invention;
FIG. 8 shows the results of the invention in Experimental example 4.
Detailed Description
Example 1:
the invention provides a biological kit for researching cell mechanical force induction, which comprises a biological glue matrix, wherein the biological glue matrix comprises the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
Example 2:
the invention relates to a biological kit for researching cell mechanical force induction, which comprises:
a glass sheet;
the coating liquid box is used for storing the first coating liquid and the second coating liquid;
a biogel substrate box for storing reagents for preparing the biogel substrate;
a perforated plate;
the lavage liquid box is used for storing a first lavage liquid and a second lavage liquid;
the activation liquid box is used for storing a first activation liquid and a second activation liquid;
and the matrix incubation liquid is used for storing the matrix incubation liquid, and comprises a matrix coating liquid and a Laminin reagent.
The biological glue matrix comprises the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
The glass sheet is a common carrier in experiments, is non-toxic and transparent, and cannot react with biomass. The perforated plate is provided with a plurality of hole sites, and can be used for simultaneously placing a plurality of glass sheets to prepare a plurality of biological glue matrixes.
Example 3:
the biogel matrix comprises the following components:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
The raw materials of the biological glue matrix are added in sequence and then mixed evenly to obtain the biological glue matrix.
In order to implement the biological kit of the above embodiments 1 to 3, the present invention also discloses a method for preparing the biological kit for studying the cytomechanical force sensing, comprising the following steps:
immersing the glass sheet in the first coating liquid for treatment for several minutes, and then taking out and airing; then immersing the glass sheet in a second coating solution for processing for a plurality of minutes, taking out and airing to obtain a coated glass sheet;
step (2), preparing a biological glue matrix;
uniformly dripping the biological glue matrix on a coated glass sheet, covering the uncoated glass sheet on the biological glue matrix, flattening the biological glue matrix, and naturally airing until the biological glue matrix is solidified; uncovering the uncoated glass sheet after solidification, and adhering the biological adhesive matrix on the coated glass sheet;
step (4), placing the coated glass sheet adhered with the biological glue matrix in the holes of the porous plate, and performing first lavage by using a first lavage liquid;
pouring the first lavage fluid, then dripping the first activation fluid for first activation, pouring the first activation fluid after the first activation, and then dripping the second activation fluid;
pouring out the second activating solution, and dropwise adding a matrix incubation solution for incubation; pouring out the matrix incubation liquid after incubation is finished, then using the second lavage liquid for carrying out second lavage, and pouring out the second lavage liquid after lavage is finished; and then packaging.
The first coating liquid acts as follows: removing organic modification components on the surface of the glass; sterilization and degerming effects;
the second coating liquid acts as follows: the coating liquid contains affinity silane, which is helpful for coupling the biological glue matrix and the inorganic glass sheet and preventing the sheet from falling off.
Lavage is used to remove uncoagulated organic compounds from the biogel matrix, which is the basis for ensuring that cells respond to the non-toxic response of the surrounding matrix.
The activating solution has the function of providing a stable polypeptide coupling environment and assisting the polypeptide protein to be modified on the surface of the matrigel, thereby realizing the adherent growth of cells.
Activating liquid 1: the pretreatment extracts harmful substances in the biogel matrix and activates corresponding chemical groups of acrylamide.
Activating liquid 2: the activating liquid 2 contains hydroxysuccinimide and ethyl carbodiimide hydrochloride, and is a coupling agent which can be combined with acrylamide groups and polypeptide proteins.
The aim of incubation is to allow the polypeptide protein to be better and more fully coupled with the glue surface and remove unreacted residual components in the activating solution.
Example 4:
in the step (1), the immersion time of the glass sheet in the first coating liquid is 5-20 min; the immersion time of the glass sheet in the second coating liquid is 5 min-20 min;
the first coating solution is 70% ethanol solution, and contains 0.1 mol/L-0.3 mol/L NaOH;
the second coating solution is 95% ethanol solution, and contains 0.5-1.5% wt of affinity silane and 3-6% wt of glacial acetic acid.
Example 5:
in the step (1), the immersion time of the glass sheet in the first coating liquid is 10 min; the immersion time of the glass sheet in the second coating liquid is 10 min;
the first coating solution is 70% ethanol solution, and contains 0.2ol/L NaOH;
the second coating solution is a 95% ethanol solution, and contains 1.2% wt of affinity silane and 5% wt of glacial acetic acid.
Example 6:
the various reagents adopted by the invention can be selected from the following reagents:
the first lavage liquid is methanol;
the second lavage liquid is serum-free DMEM solution;
the solvent of the first activating solution is double distilled water containing 10mmol/L morpholine ethanesulfonic acid and 500mmol/L NaCl;
the solvent of the second activating solution is double distilled water containing morpholine ethanesulfonic acid 10mmol/L, Nacl500mmol/L, N-hydroxysuccinimide 0.48 mol/L; n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride 0.2 mol/L.
The matrix incubation liquid comprises matrix coating liquid and a Laminin reagent; the substrate coating solution is HEPES buffer solution with the concentration of 50mmol/L, and 0.1mol/L glycine is added into the HEPES buffer solution; the concentration of the Laminin reagent dissolved in the matrix coating liquid is 50 ug/ml;
example 7:
the operating time parameters of the present invention may be selected from the following:
the first lavage time is 5 min-15 min;
the second lavage lasts for 4-6 h;
the treatment time of the first activating solution is 5-15 min;
the treatment time of the second activating solution is 5-15 min;
the incubation time of the substrate incubation liquid is 12-18 h.
Example 8: influence of material ratio of different biological glue matrixes on softness and hardness of glue
It can be seen from example 8 that when the amounts of acrylamide and methylene bisacrylamide are different, the elasticity index, i.e., the degree of softness and hardness, of the obtained biogel matrix changes, and the skilled person can select a suitable material ratio according to the required degree of softness and hardness.
Example 9:
the preparation method of the biological kit comprises the following steps:
step (1):
immersing the glass sheet in the first coating solution for processing for 10 minutes, and then taking out and airing; and then immersing the glass sheet in the second coating liquid for treatment for 10 minutes, taking out and airing to obtain the coated glass sheet.
The first coating solution is 70% ethanol solution, and contains 0.2ol/L NaOH; the second coating solution is a 95% ethanol solution, and contains 1.2% wt of affinity silane and 5% wt of glacial acetic acid.
Step (2):
preparing a biological glue matrix:
the biogel matrix comprises the following components:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
The raw materials of the biological glue matrix are added in sequence and then mixed evenly to obtain the biological glue matrix.
And (3):
uniformly dripping the biological glue matrix on the coated glass sheet, covering the uncoated glass sheet on the biological glue matrix, flattening the biological glue matrix, and naturally airing the biological glue matrix until the biological glue matrix is solidified; after solidification, uncovering the uncoated glass sheet, and adhering the biological glue matrix on the coated glass sheet.
And (4):
the coated glass plate with the adhered biogel matrix is placed in the hole of a multi-hole plate, and first lavage is carried out by using methanol as lavage solution I, wherein the lavage time of the first lavage is 5 min.
And (5):
pouring out methanol of the first lavage fluid, then dripping the first activation fluid for first activation, pouring out the first activation fluid after 10min of first activation, and then dripping the second activation fluid for treatment for 10 min.
Wherein the solvent of the first activating solution is double distilled water containing 10mmol/L morpholine ethanesulfonic acid and 500mmol/L NaCl;
wherein the solvent of the second activating solution is double distilled water containing morpholine ethanesulfonic acid 10mmol/L, Nacl500mmol/L, N-hydroxysuccinimide 0.48 mol/L; n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride 0.2 mol/L.
And (6):
pouring out the second activating solution, and dropwise adding the substrate incubation solution for incubation overnight, wherein the treatment time is 12 h; pouring out the matrix incubation liquid after incubation is finished, then using the second lavage liquid to perform lavage and cleaning for 5 hours for the second time, and pouring out the second lavage liquid after lavage is finished; and then packaging.
Wherein the substrate incubation liquid comprises a substrate coating liquid and a Laminin reagent; the substrate coating solution is HEPES buffer solution with the concentration of 50mmol/L, and 0.1mol/L glycine is added into the HEPES buffer solution; the concentration of the Laminin reagent dissolved in the matrix coating solution was 50 ug/ml.
Wherein the second lavage liquid is serum-free DMEM solution.
Experimental example: taking a biological glue matrix product for experiment
Experimental example 1:
two types of biological glue matrixes with different hardness degrees are selected, wherein the elasticity index of the soft biological glue matrix is 1000, and the elasticity index of the hard biological glue matrix is 280 kPa. And observing the microstructure of the soft biogel matrix by using a magnifying glass, and observing the combination condition of the cytoplasm matrix and the biogel matrix.
The experimental results are as follows:
an enlarged view of the soft biogel matrix microstructure is shown in figure 1;
the soft biogel matrix is bound to the cytoplasmic matrix as shown in figure 2 a;
the binding of the hard biogel matrix to the cytoplasmic matrix is shown in figure 2 b.
As can be seen from the realistic view of the soft gel 2a, corresponding cells grow on the soft gel, and the growth state of the cells is good. On a soft biological glue matrix, cells are not stimulated by mechanical force and appear blunt circles; on a hard biogel matrix, cells take on a growth-activated-like morphology under the action of mechanical forces.
Experimental example 2:
the surface gray scale, the edge flatness, the edge pressure hardness gradient and the pressure stress gradient of the soft and hard rubber edge and the core of the biological rubber matrix are detected by using an atomic force microscope, and the hardness and the roughness of the biological rubber matrix can be tested by using the atomic force microscope, so that the soft and hard conditions of the biological rubber matrix under different proportions can be well proved.
The experimental results are shown in fig. 3:
in FIG. 3, A is a gray scale image of the surface of the biogel substrate; b is the result of testing the edge flatness of the biogel substrate; c is the pressure hardness gradient of the edge area of the biological glue matrix; d is the pressure stress gradient of the edge and the core of the biological glue matrix.
A shows a gray scale image of the surface of the soft and hard gelatin biogel, which is the general reaction of the soft and hard gelatin planes, wherein the brighter the white color is, the higher the level height of the gelatin surface is;
b shows the test result of the edge flatness of the soft and hard rubber, which is the reaction of the edge flatness of the soft and hard rubber, and the redder color represents that the rubber edge is rougher;
c shows the pressure hardness gradient of the soft and hard rubber edge region, which is the reaction of the pressure of the soft and hard rubber edge region, and the redder the color represents that the rubber edge is harder;
graph D shows the pressure stress gradient of the edge and core of the soft and hard glue, and the lighter the color is, the harder the glue surface is.
Experimental example 3:
immunofluorescence staining and real-time calcium imaging test of the biological glue matrix, wherein the results of the immunofluorescence staining are shown in figure 5; the results of the real-time calcium imaging measurements are shown in fig. 6.
As shown in fig. 5, primary microglia can be well present on the biogel substrate;
the meaning of the different colors in the figure:
green: microglia Iba 1;
blue color: cell nucleus DAPI;
purple: calcium channel staining.
The experimental example is divided into three groups of biological glue matrixes with different soft and hard indexes, and as can be seen from fig. 5, the hard biological glue matrix can stimulate the microglial cell to bulge and form polarization.
Referring to fig. 6, primary cells grew well on a biocolloid matrix and were significantly sensitive to calcium channels. Soft gum, Stiff and knock-down calcium channels and activated calcium channels (Yoda) all have good cell biological reaction.
Corresponding cells are grown on the surface of the biological glue matrix, and the cellular immunofluorescence is to prove that the glue surface is non-toxic and harmless to the cells, and can realize cell staining of the matrix glue by reacting with the hardness and the morphology of different biological glue matrixes.
Real-time calcium imaging test purposes: because of the different hardness and hardness of the biological glue matrix, the harder the glue surface is, the stronger the mechanical stimulation is applied to the cell, and the more calcium signals are generated in the cytoplasm of the cell. Cells respond biologically to the stimulation of glue indirectly by means of calcium imaging.
Alphabetical meanings of the invention:
soft represents a biogel matrix with an elastic index of 1000;
stiff represents a biogel matrix with an elasticity index of about 280 kpa;
glass represents a biogel matrix with an elastic index of around 1000 kpa.
Experimental example 4: cytomechanical force sensing gene test
The test method comprises the following steps:
(1) taking primary mouse microglia;
(2) constructing three biogel matrixes with different elasticity indexes;
(3) the primary microglia cell line is evenly inoculated on the three biological glue matrixes, and protein molecules with different hardness such as: fibrillating a β (fA β) and monomeric a β (mA β), stimulating the cells.
The test purpose is as follows:
the response of the cells to additional protein molecules on the glue side of the biogel matrix of different degrees of softness was demonstrated.
And (3) testing results:
the expression of mRNA level of the mechanical force sensing protein piezo1 was examined and no significant difference was found between groups. Then, Western blot technique is used to detect protein extracted from cells of the biogel matrixes with different degrees of softness and hardness, and the response of the Piezo1 is found.
In summary, it can be seen from fig. 7 and 8 that: the biological kit can stimulate the growth of primary cells without toxicity; and successfully identifies the stimulated cells of different hardness molecular foreign bodies (fAb and mAb) and detects the expression conditions of corresponding gene mRNA and protein.
The kit can ensure that cells well live on the surface of the biological glue matrix, can respond to additional stimulation, and can realize the detection of mRNA and protein. This is another technical feature and advantage of the present kit.
Claims (10)
1. A biological kit for studying the induction of cellular mechanical forces, comprising a biogel matrix comprising the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
2. A biological kit for studying the induction of cellular mechanical forces, comprising:
a glass sheet;
the coating liquid box is used for storing the first coating liquid and the second coating liquid;
a biogel substrate box for storing reagents for preparing the biogel substrate;
a perforated plate;
the lavage liquid box is used for storing a first lavage liquid and a second lavage liquid;
the activation liquid box is used for storing a first activation liquid and a second activation liquid;
the matrix incubation liquid is used for storing the matrix incubation liquid, and comprises a matrix coating liquid and a Laminin reagent;
the biogel matrix comprises the following components:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water.
3. A biological kit for studying the cytomechanical force response according to claim 1 or 2, characterized in that: the biogel matrix comprises the following components:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
4. A preparation method of a biological kit for researching cell mechanical force induction is characterized by comprising the following steps:
immersing the glass sheet in the first coating liquid for treatment for several minutes, and then taking out and airing; then immersing the glass sheet in a second coating solution for processing for a plurality of minutes, taking out and airing to obtain a coated glass sheet;
step (2), preparing a biological glue matrix;
uniformly dripping the biological glue matrix on a coated glass sheet, covering the uncoated glass sheet on the biological glue matrix, flattening the biological glue matrix, and naturally airing until the biological glue matrix is solidified; uncovering the uncoated glass sheet after solidification, and adhering the biological adhesive matrix on the coated glass sheet;
step (4), placing the coated glass sheet adhered with the biological glue matrix in the holes of the porous plate, and performing first lavage by using a first lavage liquid;
pouring the first lavage fluid, then dripping the first activation fluid for first activation, pouring the first activation fluid after the first activation, and then dripping the second activation fluid;
pouring out the second activating solution, and dropwise adding a matrix incubation solution for incubation; pouring out the matrix incubation liquid after incubation is finished, then using the second lavage liquid for carrying out second lavage, and pouring out the second lavage liquid after lavage is finished; and then packaging.
5. The method of claim 4, wherein: the immersion time of the glass sheet in the first coating liquid in the step (1) is 5-20 min; the immersion time of the glass sheet in the second coating liquid is 5 min-20 min;
the first coating solution is 70% ethanol solution, and contains 0.1-0.3 mol/L NaOH;
the second coating solution is 95% ethanol solution, and contains 0.5-1.5% wt of affinity silane and 3-6% wt of glacial acetic acid.
6. The method of claim 4, wherein: the immersion time of the glass sheet in the first coating liquid in the step (1) is 10 min; the immersion time of the glass sheet in the second coating liquid is 10 min;
the first coating solution is 70% ethanol solution, and contains 0.2ol/L NaOH;
the second coating solution is a 95% ethanol solution, and contains 1.2% wt of affinity silane and 5% wt of glacial acetic acid.
7. The method of claim 4, wherein: the biological glue matrix is prepared from the following raw materials:
3 to 10 weight percent of acrylamide; 0.05 to 0.5 weight percent of methylene bisacrylamide;
30 mmol/L-60 mmol/L of 6-acrylamidohexanoic acid; 0.5 g/L-2 g/L of ammonium sulfate;
2 g/L-8 g/L of tetramethyl ethylenediamine; the solvent is double distilled water;
the raw materials of the biological glue matrix are added in sequence and then mixed evenly to obtain the biological glue matrix.
8. The method of claim 4, wherein: the biological glue matrix is prepared from the following raw materials:
acrylamide 3% wt; 0.1% wt of methylene bisacrylamide;
48mmol/L of 6-acrylamidohexanoic acid; 1g/L of ammonium sulfate;
4g/L of tetramethyl ethylene diamine; the solvent is double distilled water.
9. The method of claim 4, wherein:
the first lavage fluid is methanol;
the second lavage fluid is serum-free DMEM solution;
the solvent of the first activating solution is double distilled water and contains 10mmol/L morpholine ethanesulfonic acid and 500mmol/L NaCl;
the solvent of the second activating solution is double distilled water containing 10mmol/L, Nacl500mmol/L morpholine ethanesulfonic acid,
0.48mol/L of N-hydroxysuccinimide; n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride 0.2 mol/L.
The matrix incubation liquid comprises a matrix coating liquid and a Laminin reagent; the substrate coating solution is HEPES buffer solution with the concentration of 50mmol/L, and 0.1mol/L glycine is added into the HEPES buffer solution; the concentration of the Laminin reagent dissolved in the matrix coating solution was 50 ug/ml.
10. The method of claim 4, wherein:
the first lavage time is 5 min-15 min;
the second lavage time is 4-6 h;
the treatment time of the first activating solution is 5-15 min;
the treatment time of the second activating solution is 5-15 min;
the incubation time of the substrate incubation liquid is 12-18 h.
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| CN103898055A (en) * | 2014-01-20 | 2014-07-02 | 复旦大学附属中山医院 | Establishment method of in-vitro cell culture platform with different matrix hardnesses |
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| CN103898055A (en) * | 2014-01-20 | 2014-07-02 | 复旦大学附属中山医院 | Establishment method of in-vitro cell culture platform with different matrix hardnesses |
| CN106687149A (en) * | 2014-07-09 | 2017-05-17 | 路博润先进材料公司 | Hydrogel compositions |
| US20180216063A1 (en) * | 2015-07-22 | 2018-08-02 | The Johns Hopkins University | Three-dimensional vascular network assembly from induced pluripotent stem cells |
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