CN114990057B

CN114990057B - Method for separating and culturing intramuscular fat cells of buffalo fetuses and inducing differentiation into fat

Info

Publication number: CN114990057B
Application number: CN202210697735.9A
Authority: CN
Inventors: 黄洁萍; 朱锐锐; 石德顺; 李瑞瑞; 冯偞
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2024-04-26
Anticipated expiration: 2042-06-20
Also published as: CN114990057A

Abstract

The invention relates to the field of cell engineering and tissue engineering, in particular to a method for separating and culturing intramuscular fat cells of buffalo fetuses and inducing differentiation into fat. The method comprises the following steps: taking the longus muscle of the buffalo embryo cattle back, and cleaning and sterilizing; dividing the longissimus tissue of buffalo embryo and cattle back; adding 0.2% collagenase I for digestion; adding complete medium to stop digestion; centrifuging the cells which terminate digestion, and discarding the supernatant to obtain a cell pellet; purifying cells by differential adherence; culturing the purified cells in an incubator with 5% CO ₂ at 37 ℃ for 2 days, and changing the complete culture medium once; after the cell density reaches about 80%, the induction medium is replaced, the maintenance medium is replaced after the induction is carried out for 2 days, and then the maintenance medium is replaced every 2 days. The intramuscular fat cells obtained by the buffalo fetus obtained by the method have higher activity and capacity of adipogenic differentiation.

Description

Method for separating and culturing intramuscular fat cells of buffalo fetuses and inducing differentiation into fat

Technical Field

The invention relates to the field of cell engineering and tissue engineering, in particular to a method for separating and culturing intramuscular fat cells of buffalo fetuses and adipogenic induced differentiation of the same.

Background

The current research shows that the nutrition value of buffalo meat is no more than that of yellow beef. Secondly, the current beef supply is not in demand. Therefore, the method has important significance for fully utilizing rich buffalo resources to relieve the supply and demand pressure of domestic beef. The flavor, taste and juiciness of buffalo are seriously affected by poor intramuscular deposition capability, so that the improvement of intramuscular fat deposition capability of buffalo is a problem to be solved. The buffalo fetal intramuscular fat cells are separated in vitro, cultured, induced and differentiated, and the buffalo intramuscular fat deposition is simulated in vitro, so that a referential meaning is provided for improving the meat quality of buffalo and analyzing the molecular mechanism of buffalo intramuscular fat deposition.

No technology for in vitro culture, induction and research of intramuscular adipocyte cells of other animals in fetal period and the technology for inducing adipogenesis is reported.

The invention patent application with the application publication number of CN109679898A discloses an improved method for inducing bovine intramuscular fat cells to perform adipogenic differentiation, which comprises the steps of culturing bovine intramuscular fat cells in DMEM/F12 culture medium containing 10% fetal bovine serum, and adding an induced differentiation agent for 2 days after the confluence of the bovine intramuscular fat cells reaches 100%. The composition of the induced differentiation agent is as follows: 3-isobutyl-1-methylxanthine: 0.5mM, dexamethasone: 1 μm, insulin: 10 μg/mL, rosebeliazone: 2. Mu. Mol/L.

Disclosure of Invention

The invention aims to provide a method for obtaining intramuscular fat cells of buffalo with better activity for isolated culture and induced adipogenic differentiation, and the obtained intramuscular fat cells have higher adipogenic differentiation capability.

The aim of the invention is achieved by the following technical scheme:

a method for separating, culturing and adipogenic inducing differentiation of buffalo fetal intramuscular fat cells comprises the following steps:

Step 1: and (3) disinfection: firstly, the epidermis of the buffalo embryo cattle is cleaned, the epidermis is removed, the longus muscle of the cattle dorsum of the buffalo embryo is taken, and the longus muscle tissue of the cattle dorsum is disinfected under the aseptic condition. Intramuscular fat content is one of the key indicators for evaluating beef quality, and the measurement criteria for intramuscular fat content (i.e., the level of intramuscular fat deposition) are: the longissimus dorsi cross section was taken and compared with a comparison card, or the longissimus dorsi was taken and the crude fat content was determined. Thus, the invention also uses the longus muscle of the back of the cow as a material to isolate intramuscular fat cells.

Specifically, the washing is to wash the buffalo embryo cattle skin by using 3 times of blue/streptomycin phosphate buffer salt solution, remove the skin and take the longus muscle of the buffalo embryo cattle back. The phosphate buffer salt solution of 3 times of penicillin/streptomycin is phosphate buffer salt solution added with 3 percent of penicillin/streptomycin, and the blue/streptomycin solution is a mixed solution prepared by 100U/mL penicillin and 0.1mg/mL streptomycin.

The sterilization is to soak the longissimus dorsum tissue of the cattle in sequence for 2 minutes by using a phosphate buffer solution which contains 1 percent, 2 percent and 3 percent of blue/chain mycin which is preheated under the aseptic condition, soak the longissimus dorsum tissue in 75 percent of alcohol for 30 seconds, and soak the longissimus dorsum tissue in sequence for 2 minutes by using the phosphate buffer solution which contains 3 percent, 2 percent and 1 percent of blue/chain mycin which is preheated.

Step 2: segmentation: dividing the longest muscle tissue blocks of the beef dorsum of the disinfected buffalo embryo; specifically, the sterilized longissimus muscle tissue blocks of buffalo embryo and cattle back can be divided into 1-2 mm ³ by scissors.

Step 3: digestion: the divided tissue pieces were digested by adding 0.2% collagenase I. Specifically, 1-2 mm ³ tissue blocks are transferred into a 15mL centrifuge tube by using a 1mL pipette, 0.2% collagenase I which is 2 times of the volume of the tissue blocks is added, and the tissue blocks are digested for 90-120 min on a constant temperature shaking table at 37 ℃ until flocculence appears.

Step 4: and (3) screening: adding complete culture medium to the digested tissue to terminate digestion; specifically, digestion was terminated by adding DMEM medium containing 20% fetal bovine serum and 1% diabody to the digested tissue in an equivalent volume of 0.2% collagenase I.

Step 5: and (3) centrifuging: centrifuging the cells which terminate digestion, and discarding the supernatant to obtain a cell pellet; specifically, the cells which have stopped digestion are transferred to a 50mL centrifuge tube through a 200 mesh cell sieve, and centrifuged at 1500rpm for 10min.

Step 6: purifying: the centrifuged cells are resuspended in DMEM medium containing 20% fetal calf serum and 1% diabody, and the cells are purified by differential adherence;

The differential adherence method comprises the following steps: culturing cells in an incubator at 37deg.C and 5% CO ₂ for 1 hr, discarding culture medium containing non-adherent cells, washing with 1 times of blue/streptomycin phosphate buffer solution for 2 times, and adding DMEM culture medium containing 20% foetal calf serum and 1% double antibody into the cells; the phosphate buffer solution of 1 times of blue/streptomycin is phosphate buffer solution added with 1% of blue/streptomycin.

Step 7: culturing: the purified cells were cultured in an incubator at 37℃with 5% CO ₂, and the DMEM medium containing 20% fetal bovine serum and 1% diabody was changed every 2 days. After the cell density reaches about 80%, the induction culture medium is replaced, the maintenance culture medium is replaced after the induction is carried out for 2 days, and then the maintenance culture medium is replaced every two days for 6-8 days.

The components of the induced differentiation agent in the induction medium are as follows: 0.5mM 3-isobutyl-1-methylxanthine, 1. Mu.M dexamethasone, 10. Mu.g/mL insulin and 1. Mu.M rosiglitazone; the components of the maintenance differentiation agent in the maintenance medium: 10 μg/mL insulin and 1 μM rosiglitazone.

The beneficial effects of the invention are as follows:

the intramuscular fat cells obtained by the buffalo fetus obtained by the method have higher activity and capacity of adipogenic differentiation.

The method for inhibiting the intramuscular fat cells of the buffalo fetuses by contact does not need to adopt a method that the density is close to 100 percent and the contact is inhibited for 2 days, and the better induction effect can be achieved when the density reaches 80 percent.

Drawings

Fig. 1: morphological observation of buffalo intramuscular adipocytes. FIG. 1A shows purified buffalo intramuscular fat cells after 2 days of culture; FIG. 1B shows the purified buffalo intramuscular fat cells after 6 days of culture.

Fig. 2: induced culture of buffalo intramuscular adipocytes for 0, 2,4, 6, 8, 10 days of the cell conditions of example 1. In the figure, "Day" means "Day".

Fig. 3: detection of expression levels of adipogenic marker Gene fatty acid binding protein 4 (FATTY ACID-binding protein, FABP 4), peroxisome proliferator-activated receptor gamma (peroxisome proliferators-ACTIVATED RECEPTORS gamma, PPARgamma or PPARG), and enhancer binding protein alpha (CCAAT/enhancer binding proteins alpha, C/EBP alpha). The "d" on the abscissa in the figure represents "day".

Fig. 4: oil red O staining patterns of uninduced cells and cells after induction for 10 days in example 1. The left side of FIG. 4 shows the induced cells, and the right side of FIG. 4 shows the cells after 10 days of induction.

Fig. 5: the 80% density direct induction of buffalo intramuscular adipocytes of example 1 and the 100% density contact inhibition of example 2 induced conditions after 2 days. In the figure, "day" means "day".

Fig. 6: absorbance of uninduced cells and 10 day post-induction cell oil red O staining in example 1. The "d" on the abscissa in the figure represents "day".

Fig. 7: triglyceride levels in cells after 0, 2, 6, 10 days of induction in example 1. "day" on the abscissa in the figure indicates "day".

Fig. 8: western blot was used to detect intracellular levels of PPARG protein during different induction periods. In the figure, "Day" means "Day".

Detailed Description

The following detailed description of the present invention is provided to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of the present invention.

Experimental reagent: type I collagenase (Solarbio), high sugar medium (Hyclone), fetal Bovine Serum (FBS) (holly), green/streptomycin (Hyclone), trypsin (Hyclone); bovine insulin (Sigma), dexamethasone (Sigma), 3-isobutyl-1-methylxanthine (Sigma), rosiglitazone (Sigma), oil red O powder (Sigma), triglyceride assay kit (priril), quantitative enzyme, reverse transcriptase, protein Marker (Vazyme), phosphate buffered saline (Phosphate buffer saline, PBS), beta-actin rat anti-purchased from Biyunnan, PPARG rabbit anti-purchased from Abisxin, RIPA tissue/cell lysate and PMSF purchased from Soxhoba.

The 75% alcohol in the examples represents 75% by volume of ethanol in the alcohol.

The double antibody is a green/streptomycin solution, and is a mixed solution prepared from 100U/mL penicillin and 0.1mg/mL streptomycin.

Phosphate buffer solution of 1 or 3 times of blue/streptomycin is prepared by adding 1% or 3% of blue/streptomycin by volume percent into phosphate buffer solution.

The phosphate buffer solution preheated by 1%, 2% and 3% of cyan/streptomycin is the phosphate buffer solution preheated by 1%, 2% and 3% of cyan/streptomycin by volume percentage.

0.2% Collagenase I refers to: 10mLDMEM, the mass of collagenase I is 0.02g.

The DMEM medium containing 20% of fetal calf serum and 1% of diab is the DMEM medium containing 20% of fetal calf serum and 1% of diab by volume.

The induction medium is prepared by adding an induction differentiation agent into DMEM medium containing 20% of fetal calf serum and 1% of diabody.

The maintenance medium is a DMEM medium containing 20% fetal bovine serum and 1% diabody, and a maintenance differentiation agent is added.

Oil red O mother liquor: 0.25g of oil red O powder was dissolved in 50mL of isopropanol.

By 4% paraformaldehyde is meant that the paraformaldehyde solution comprises 4% by volume paraformaldehyde.

60% Isopropyl alcohol means that the isopropyl alcohol solution contains 60% by volume of isopropyl alcohol.

Example 1

Step 1: and (3) disinfection: the epidermis of buffalo embryo cattle is firstly washed by phosphate buffer salt solution of 3 times of green/streptomycin, the epidermis is removed, and the longest muscle of the back of buffalo embryo cattle is taken. Under the aseptic condition, the longest muscle tissue of the back of the cattle is soaked in the phosphate buffer salt solution which contains 1 percent, 2 percent and 3 percent of the blue/chain mycin for 2 minutes in sequence, is soaked in the alcohol of 75 percent for 30 seconds, and is soaked in the phosphate buffer salt solution which contains 3 percent, 2 percent and 1 percent of the blue/chain mycin for 2 minutes in sequence.

Step 2: segmentation: the sterilized longissimus muscle tissue blocks of buffalo embryo and cattle back are divided into 1-2 mm ³ by scissors.

Step 3: digestion: transferring 1-2 mm ³ tissue blocks into a 15mL centrifuge tube by using a 1mL pipetting gun, adding 0.2% collagenase I which is 2 times the volume of the tissue blocks, and digesting for 90-120 min on a constant temperature shaking table at 37 ℃ until flocculence appears.

Step 4: and (3) screening: digestion was terminated by adding the digested tissue to DMEM medium containing 20% fetal bovine serum and 1% diabody in an equivalent volume to 0.2% collagenase I.

Step 5: and (3) centrifuging: the cells which have stopped digestion are transferred through a 200 mesh cell sieve to a 50mL centrifuge tube and centrifuged at 1500rpm for 10min, and the supernatant is discarded to give a cell pellet.

Step 6: purifying: the centrifuged cells were resuspended in DMEM medium containing 20% fetal bovine serum and 1% diabody and purified by differential adherence.

The differential adherence method comprises the following steps: culturing the resuspended cells in an incubator at 37 ℃ and 5% CO ₂ for 1h, discarding the culture medium containing non-adherent cells, washing 2 times with 1 times of blue/streptomycin phosphate buffer solution, and then adding DMEM culture medium containing 20% fetal calf serum and 1% diabody into the cells for continuous culture; the phosphate buffer solution of 1 times of blue/streptomycin is phosphate buffer solution added with 1% of blue/streptomycin.

Step 7: culturing: purified cells were cultured in an incubator at 37℃with 5% CO ₂, and the DMEM medium containing 20% fetal bovine serum and 1% diabody was changed every 2 days. After the cell density reaches about 80%, the induction culture medium is replaced, the maintenance culture medium is replaced after the induction is carried out for 2 days, and then the maintenance culture medium is replaced every two days for 6-8 days.

Example 2

Step 1: and (3) disinfection: the epidermis of buffalo embryo cattle is firstly washed by phosphate buffer salt solution of 3 times of green/streptomycin, the epidermis is removed, and the longest muscle of the back of buffalo embryo cattle is taken. Under aseptic condition, the longest muscle tissue of the back of the cattle is soaked in phosphate buffer salt solution which contains 1%, 2% and 3% of blue/chain mycin for 2min, is soaked in 75% of alcohol for 30s, and is soaked in PBS which contains 3%, 2% and 1% of blue/chain mycin for 2min.

Step 2: segmentation: the sterilized longus muscle tissue blocks of the cattle back are divided into 1-2 mm ³ by scissors.

Step 3: digestion: transferring 1-2 mm ³ tissue blocks into a 15mL centrifuge tube by using a 1mL pipetting gun, adding 0.2% collagenase I which is 2 times the volume of the tissue blocks, and digesting for 90-120 min (until flocculence appears) on a constant temperature shaking table at 37 ℃.

Step 7: culturing: purified cells were cultured in an incubator at 37℃with 5% CO ₂, and the DMEM medium containing 20% fetal bovine serum and 1% diabody was changed every 2 days. After the cell density reaches 100% and the contact inhibition is carried out for 2 days, the induction culture medium is replaced, the maintenance culture medium is replaced after the induction is carried out for 2 days, and then the maintenance culture medium is replaced every two days for 6-8 days.

1. Morphological observation of intramuscular fat cells of buffalo fetuses

After the cells centrifuged in step 6 of example 1 were resuspended in DMEM medium containing 20% fetal bovine serum and 1% diabody, the cells were pellet-shaped and in suspension.

After the purified irregular triangle and fusiform cells obtained in the step 6 of the example 1 are continuously cultured for 2 days in an incubator with 5% CO ₂ at 37 ℃, intramuscular fat cells start to stretch, and form fibers when the shuttles appear, as shown in fig. 1A, the cells are vigorous, the cell clone density can reach 100% after continuous culture for 4 days, as shown in fig. 1B, and the cells can be passaged.

(1) Lipid-forming induced differentiation of buffalo fetal intramuscular adipocytes

As can be seen from fig. 2, the cells after induction for 2 days in example 1 were rounded, and the adipogenesis thereof was more and more apparent as the induction time of the intramuscular adipocytes was increased.

Total RNA of cells (cells induced for 0,2, 4, 6, 8, 10 days) was extracted by TRIzol method, RNA was inverted into cDNA by using a Reprandial reverse transcription kit, qRT-PCR experiments were performed by using a quantitative enzyme of Reprandial, and primers used for qRT-PCR are shown in Table 1. Data processing and analysis was then performed using GRAPHPAD PRISM f.

As shown in FIG. 3, the expression level of adipogenic marker genes (FABP 4, PPARG and C/EBP. Alpha.) was significantly increased by inducing differentiation of intramuscular adipocytes of buffalo fetuses.

TABLE 1qRT-PCR primers

2. Detection of cell induction effect by oil red O

Cells to be stained with oil red O (cells induced for 0 day, 10 days in examples 1, 2) were discarded, washed 3 times with PBS, fixed for 30min with 4% paraformaldehyde, prepared with oil red O working solution (PBS: oil red O mother liquor (volume ratio) =2:3), left to stand for 20min, filtered through a 0.25 μm filter membrane, discarded with 4% paraformaldehyde, washed with 60% isopropanol, discarded with 60% isopropanol, added with oil red O working solution, stained for 30min, discarded with oil red O working solution, washed 3 times with PBS, and photographed with a microscope.

As a result, as shown in FIG. 4, lipid droplets were stained red with oil red O, and cells after 10 days of induction in example 1 were much more than lipid droplets in uninduced cells.

FIG. 5 illustrates that 80% density direct induction of buffalo intramuscular fat cells of example 1 resulted in mature macrofat droplets, while 100% density contact inhibition of example 2 was not good after adipogenic induction for 2 days.

Measurement of stained oil red O stained cells by enzyme-labeling instrument:

The cells of example 1, which had been stained with oil red O, were washed with 100% isopropanol, added to a 96-well plate using a pipette at 100. Mu.L per well, and the OD was measured at a wavelength of 510nm using an ELISA reader. As a result, as shown in FIG. 6, the absorbance of the cells induced for 10 days in example 1 was significantly higher than that of the uninduced cells at 510 nm.

3. Induction of triglyceride determination in cells

The medium in the cells induced for 0, 2,6, 10 days in example 1 was discarded, washed 2 times with PBS, the cells were collected by digestion with pancreatin, centrifugation and then lysed, and 100. Mu.L of lysate was added to the cells of each well, and the mixture was allowed to stand at room temperature for 10min. Transferring proper amount of lysate into a 1.5mL centrifuge tube for triglyceride measurement, and storing the rest lysate in BCA method protein quantification kit (Biyun day) at-20deg.C. The lysate used for triglyceride measurement was heated at 70℃for 10min, and flocculent precipitate may appear when the tissue amount was large. Centrifugation at 2000rpm for 5min at room temperature, the supernatant was used for enzymatic identification.

As shown in fig. 7, the induced differentiation of buffalo fetal intramuscular adipocytes significantly increased intracellular triglyceride levels.

4. Western blot detection of intracellular PPARG protein levels at different induction periods

Buffalo fetal intramuscular adipocytes induced for 0, 2, 6, 10 days after induction of example 1 were collected. And extracting buffalo fetus intramuscular fat cell proteins in different induction periods by using RIPA tissue/cell lysate and PMSF. Running electrophoresis by using 10% SDS-PAGE, transferring film, shaking table sealing at room temperature by 5% skimmed milk powder for 1h, incubating overnight at the temperature of the primary antibody at 4 ℃, shaking table incubating for 1h at the temperature of the secondary antibody, and developing color. The results are shown in fig. 8, which induced 2 days higher levels of PPARG protein than other periods. This means that when culturing in step 7, the operation of changing the induction medium and changing the maintenance medium after the induction for 2 days after the cell density reaches about 80%, and that excellent induction effect can be obtained for the buffalo fetal intramuscular fat cells.

The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention, so that all equivalent changes or modifications of the structure, characteristics and principles described in the claims should be included in the scope of the present invention.

Claims

1. A method for separating, culturing and adipogenic inducing and differentiating intramuscular fat cells of buffalo fetuses, which is characterized by comprising the following steps:

Step 1: and (3) disinfection: firstly, cleaning the epidermis of the buffalo embryo cattle, removing the epidermis, taking the longus muscle of the cattle dorsum of the buffalo embryo, and sterilizing the longus muscle tissue of the cattle dorsum under the aseptic condition;

step 2: segmentation: dividing the longest muscle tissue blocks of the beef dorsum of the disinfected buffalo embryo;

step 3: digestion: adding 0.2% collagenase I into the segmented tissue blocks for digestion;

step 4: and (3) screening: adding complete culture medium to the digested tissue to terminate digestion;

Step 5: and (3) centrifuging: centrifuging the cells which terminate digestion, and discarding the supernatant to obtain a cell pellet;

Step 6: purifying: re-suspending the centrifuged cells in a complete culture medium, and purifying the cells by a differential adherence method;

Step 7: culturing: culturing the purified cells in an incubator with 5% CO ₂ at 37 ℃ for 2 days, and changing the complete culture medium once; after the cell density reaches 80%, changing the induction culture medium, inducing for 2 days, changing the maintenance culture medium again, and changing the maintenance culture medium every 2 days;

The induction culture medium is a complete culture medium added with an induction differentiation agent, and the induction differentiation agent in the induction culture medium comprises the following components: 0.5 mM 3-isobutyl-1-methylxanthine, 1. Mu.M dexamethasone, 10. Mu.g/mL insulin and 1. Mu.M rosiglitazone;

The maintenance medium is a complete medium added with a maintenance differentiation agent, and the maintenance differentiation agent in the maintenance medium comprises the following components: 10. mu.g/mL insulin and 1. Mu.M rosiglitazone.

2. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

In the step 1, the buffalo embryo bovine epidermis is cleaned by adopting 3 times of blue/streptomycin phosphate buffer salt solution, wherein the 3 times of blue/streptomycin phosphate buffer salt solution is a mixed solution prepared by adding 3% of blue/streptomycin into phosphate buffer salt solution, and the blue/streptomycin solution is 100U/mL penicillin and 0.1mg/mL streptomycin.

3. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

In the step 1, the cattle dorsum longissimus tissues are soaked for 2 min sequentially by using a phosphate buffer salt solution containing 1%, 2% and 3% of green/streptomycin preheating, soaked for 30s by using 75% of alcohol, and soaked for 2 min sequentially by using a phosphate buffer salt solution containing 3%, 2% and 1% of green/streptomycin preheating.

4. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

The complete medium is DMEM medium containing 20% fetal bovine serum and 1% diabody.

5. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

Step 2 is to divide the sterilized longus muscle tissue blocks of the cattle back into 1-2 mm ³.

6. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

Step 3 is to add collagenase I with the volume of 0.2% which is 2 times of the volume of the tissue blocks into the segmented tissue blocks, and digest the tissue blocks on a constant temperature shaking table with the temperature of 37 ℃ to 90 min-120 min.

7. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 6, wherein,

In step 4, the volume of complete medium added was the same as the volume of 0.2% collagenase I added in step 3.

8. The method for isolated culture and adipogenic induced differentiation of intramuscular fat cells of buffalo fetus according to claim 1, wherein,

The differential adherence method in the step 6 is as follows: culturing cells in an incubator at 37deg.C and 5% CO ₂ for 1 h, discarding culture medium containing non-adherent cells, washing with 1 times of blue/streptomycin phosphate buffer solution for 2 times, and adding DMEM culture medium containing 20% foetal calf serum and 1% double antibody into the cells;

The phosphate buffer solution of 1 times of blue/streptomycin is phosphate buffer solution added with 1% of blue/streptomycin.