A visual detection technique for autophagy activity of cotton callus cells

Abstract

The application discloses a kind of cotton callus autophagy activity visual detection technology, first using pretreatment buffer to incubate callus, then using PBS buffer transition, using MDC dyeing liquid is dyed, prepare cell suspension, observation.The application increases the level of cell autophagy by pretreatment induction, significantly improves the fluorescence staining intensity, improves the contrast of specific dyed autophagosome and background, so that autophagy in cell is more easily observed;Meanwhile, the pretreatment buffer has the effect of maintaining osmotic pressure, keeping liquid environment pH stable and providing basic nutrition, which can significantly improve the stability of staining.The application uses PBS buffer to wash, which can avoid over-staining and reduce the degree of background clutter.The staining step of the application is more simple, with clear staining levels and clear cell contrast, so that the autophagy phenomenon in plant somatic embryogenesis process is efficiently visualized.

Description

TECHNICAL FIELD

[0001] The present application relates to a cotton callus autophagy activity visual detection technology, belonging to the field of biological staining. BACKGROUND

[0002] Under the conditions of nutrient deficiency or stress, eukaryotes recycle inactivated proteins, damaged cell structures and organelles in cells through autophagy pathways. Autophagy is an important material recycling pathway in eukaryotic cells. The process first wraps the recycled material in a membrane, and then transports it to the lysosome, vacuole for degradation, or is secreted outside the cell to provide essential raw materials for cell reconstruction, regeneration and repair. The key step of autophagy induction is the formation of autophagosomes. A typical autophagosome is a closed, spherical structure formed by wrapping part of the cytoplasm, degraded organelles (such as mitochondria, endoplasmic reticulum, ribosomes), proteins, etc. in a membrane (mostly double-layered, sometimes multi-layered or single-layered). The diameter is about 0.5-1.5 μm. In the past few decades, research based on the morphogenesis of autophagosomes to detect autophagy processes has made significant progress, and autophagosomes have been observed in the ultrastructure of various organisms such as yeast, fruit flies, mice and humans. Interestingly, recent studies have found that complex autophagy phenomena also exist in plant somatic embryogenesis, but due to the characteristics of high vacuolization, loose structure and easy breakage of callus cells, it is relatively difficult to detect autophagy activity in plant somatic embryogenesis.

[0003] Dansylcadaverine (MDC) as an acidophilic dye can specifically label autophagosomes by ion trapping and specific binding with membrane lipids. Due to this property, MDC is commonly used as an indicator for autophagosome detection, with an excitation filter wavelength of 355-380 nm and a blocking filter wavelength of 512-530 nm. The traditional method first uses rapamycin or chloroquine for starvation treatment to induce autophagy, and then dilutes the MDC mother liquor with distilled water to prepare a working solution for cell staining. However, this method has the following defects: high induction cost, short staining solution life, sensitivity to the environment, unstable staining results, and difficulty in capturing fluorescent staining results. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a cotton callus autophagy activity visual detection technology. Compared with the traditional MDC staining method, the present application has the following advantages: the cell autophagy level is increased by pretreatment induction, the fluorescence staining intensity is significantly improved, the contrast of the specifically stained autophagosome and the background is improved, and the autophagy in the cell is more easily observed; at the same time, the pretreatment buffer has the functions of maintaining osmotic pressure, keeping the pH of the liquid environment stable, and providing basic nutrients, which can significantly improve the stability of the staining; the staining solution is easy to prepare, the staining steps are simple, the fluorescence quenching after staining is slow, and it is convenient for observation and photography.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is: a cotton callus autophagy activity visual detection technology, comprising the following steps:

[0006] (1) Take the callus samples to be detected at the same period, wash them, then immerse the samples in the pretreatment buffer, and incubate to induce the occurrence of cell autophagy;

[0007] (2) Discard the pretreatment buffer, add PBS buffer, and transition;

[0008] (3) Discard the PBS buffer, immerse the samples in the MDC staining solution, and stain;

[0009] (4) Discard the MDC staining solution, wash it again, add PBS buffer, and blow and disperse to obtain a callus cell suspension;

[0010] (5) Prepare a callus cell temporary slide, observe the green fluorescence using a fluorescence microscope, and analyze the autophagy activity of the callus cells.

[0011] Further, the preparation method of 1 liter of PBS buffer is as follows: 8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 are dissolved in 800mL of distilled water, the pH value is adjusted to 7.4 with an HCl solution, and finally distilled water is added to make up to 1L.

[0012] Further, the pretreatment buffer comprises the following components at the following concentrations: 234.48mM of NaCl, 10.66mM of KCl, 52.38mM of NaHCO3, 2.02mM of NaH2PO4, and 11.12mM of D-Glucose.

[0013] Further, the preparation method of the MDC staining solution is as follows: an appropriate amount of MDC mother liquor (1000X) is diluted with PBS buffer at a volume ratio of 1:1000 to obtain the MDC staining solution (1X).

[0014] Further, the incubation temperature in step (1) is 37℃, and the time is 30-60min.

[0015] Further, the transition temperature in step (2) is 37℃, and the time is 10 min.

[0016] Further, the staining condition in step (3) is 37℃, dark staining for 30-60 min.

[0017] Further, the reagent used for washing is PBS buffer.

[0018] Beneficial effects:

[0019] The autophagy activity of normally cultured cells is very low, and is not suitable for observation, so an autophagy inducer is needed for artificial intervention and regulation of autophagy. The pretreatment buffer of the present application belongs to the phosphate buffer commonly used in cell separation or culture, and the main components are glucose, NaHCO3, NaCl and KCl, which have the functions of maintaining salt balance, adjusting pH and maintaining biological active substances, and can also induce autophagy to occur, and the operation is simple and the cost is low. At the same time, K + and Na + in the pretreatment buffer can adjust the permeability and tension, maintain the integrity of the cell membrane and internal structure, and not easily destroy the structure and biological properties of the cells. Before MDC staining, the sample is treated with the pretreatment buffer, which can improve the intensity of the staining.

[0020] In addition, the use of PBS buffer for washing can avoid over-staining and reduce the degree of background clutter. Resuspension with PBS buffer can avoid excessive crowding of cells, which is conducive to the observation and subsequent quantification of autophagy. Using PBS buffer as a solvent has the functions of salt balance, adjustable suitable pH buffering, and can dissolve and protect reagents. The staining step of the present application is more concise, the staining levels are clear, and the contrast of the cells is clear, which makes the autophagy phenomenon in the process of plant somatic embryogenesis highly visualized. BRIEF DESCRIPTION OF DRAWINGS

[0021] Fig. 1 The staining effect microscopic picture of MDC staining provided for Example 1.

[0022] Fig. 2 The staining effect microscopic picture of MDC staining provided for Example 2.

[0023] Fig. 3 The staining effect microscopic picture of MDC staining provided for Example 3.

[0024] Fig. 4 The staining effect microscopic picture of MDC staining provided for Comparative Example 1.

[0025] Fig. 5 The staining effect microscopic picture of MDC staining provided for Comparative Example 2.

[0026] Fig. 6 The staining effect microscopic picture of MDC staining provided for Comparative Example 3. DETAILED DESCRIPTION

[0027] The specific embodiments of the present application are further described in detail below with reference to the following examples.

[0028] Preparation of reagents:

[0029] The preparation method of each liter of PBS buffer solution is as follows: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 are dissolved in 800 mL of distilled water, and the pH value is adjusted to 7.4 with an HC1 solution, and finally distilled water is added to make up to 1 L.

[0030] The pretreatment buffer solution comprises the following components at the following concentrations: 234.48 mM of NaCl, 10.66 mM of KCl, 52.38 mM of NaHCO3, 2.02 mM of NaH2PO4, and 11.12 mM of D-glucose, and the solvent is distilled water.

[0031] The preparation method of the MDC staining solution is as follows: an appropriate amount of MDC mother liquor (1000X) is diluted with PBS buffer solution at a volume ratio of 1:1000 to obtain the MDC staining solution (1X).

[0032] Example 1: A visual detection technology for autophagy activity of cotton callus cells

[0033] (1) A cotton callus tissue sample (about 0.05 g) to be detected at the same period is placed in a test tube, washed once with PBS buffer solution, and then 2 mL of pretreatment buffer solution is added to immerse the sample, and the sample is incubated at 37°C in a cell incubator for 30 min to induce autophagy to occur;

[0034] (2) The pretreatment buffer solution is discarded, 2 mL of PBS buffer solution is added to immerse the sample, and the sample is incubated at 37°C in a cell incubator for 10 min;

[0035] (3) The PBS buffer solution is discarded, 1 mL of MDC staining solution is added to immerse the sample, and the sample is stained at 37°C in a cell incubator in the dark, and the staining time can be appropriately adjusted within the range of 30-60 min according to the actual staining effect;

[0036] (4) The MDC staining solution is discarded, and the sample is washed 3 times with PBS buffer solution, each time using 2 mL of PBS buffer solution; the PBS buffer solution is discarded, and finally 1 mL of PBS buffer solution is added, and the sample is gently blown and dispersed to obtain a callus cell suspension;

[0037] (5) Take a drop of cell suspension on a glass slide, cover with a cover glass, remove the excess PBS buffer, and place the prepared cell temporary slide on the fluorescence microscope operating table, excite under the microscope with 355-380 nm excitation light, observe green fluorescence, and analyze cell autophagy activity.

[0038] Example 2: A visual detection technology for cotton callus cell autophagy activity

[0039] (1) Take the same period of cotton callus tissue block samples (about 0.05g) to be detected into test tubes, wash once with PBS buffer, then add 2mL of pre-treatment buffer to immerse the sample, and incubate at 37℃ in a cell incubator for 45min to induce cell autophagy;

[0040] (2) Discard the pre-treatment buffer, add 2mL of PBS buffer to immerse the sample in each tube, and transition at 37℃ in a cell incubator for 10min;

[0041] (3) Discard the PBS buffer, add 1mL of MDC staining solution to immerse the sample in each tube, and stain at 37℃ in a cell incubator in the dark, the staining time can be appropriately adjusted within the range of 30-60min according to the actual staining effect;

[0042] (4) Discard the MDC staining solution, wash 3 times with PBS buffer, each time using 2mL of PBS buffer; discard the PBS buffer, and finally add 1mL of PBS buffer, gently blow and disperse to obtain a callus cell suspension;

[0043] (5) Take a drop of cell suspension on a glass slide, cover with a cover glass, remove the excess PBS buffer, and place the prepared cell temporary slide on the fluorescence microscope operating table, excite under the microscope with 355-380 nm excitation light, observe green fluorescence, and analyze cell autophagy activity.

[0044] Example 3: A visual detection technology for cotton callus cell autophagy activity

[0045] (1) Take the same period of cotton callus tissue block samples (about 0.05g) to be detected into test tubes, wash once with PBS buffer, then add 2mL of pre-treatment buffer to immerse the sample, and incubate at 37℃ in a cell incubator for 60min to induce cell autophagy;

[0046] (2) Discard the pre-treatment buffer, add 2mL of PBS buffer to immerse the sample in each tube, and transition at 37℃ in a cell incubator for 10min;

[0047] (3) Discard the PBS buffer solution, add 1 mL of MDC staining solution to each tube to immerse the sample, and perform staining in a cell incubator at 37°C in the dark. The staining time can be appropriately adjusted in the range of 30-60 min according to the actual staining effect;

[0048] (4) Discard the MDC staining solution, wash with PBS buffer solution for 3 times, each time using 2 mL of PBS buffer solution; discard the PBS buffer solution, and finally add 1 mL of PBS buffer solution, gently blow and disperse to obtain a callus cell suspension;

[0049] (5) A drop of the cell suspension is coated on a glass slide, covered with a cover glass, the excess PBS buffer solution is removed, and the prepared cell temporary mounting is placed on the operation table of a fluorescence microscope, excited under the microscope with 355-380 nm excitation light, green fluorescence is observed, and the autophagy activity of the cells is analyzed.

[0050] Comparative Example 1: A visual detection technology for autophagy activity of cotton callus cells

[0051] The method of the present comparative example is basically the same as that of Example 3, except that distilled water is used instead of the pretreatment buffer solution.

[0052] Comparative Example 2: A visual detection technology for autophagy activity of cotton callus cells

[0053] The method of the present comparative example is basically the same as that of Example 3, except that the cells are not treated with the pretreatment buffer solution before staining.

[0054] Comparative Example 3: A visual detection technology for autophagy activity of cotton callus cells

[0055] The method of the present comparative example is basically the same as that of Example 3, except that a mixture of rapamycin solution (300 nM) and chloroquine solution (10 μM) at a volume ratio of 1:1 is used instead of the pretreatment buffer solution.

[0056] Examples 1-3 of the present application use the pretreatment buffer solution as the experimental group, and Comparative Examples 1-3 are used as the control group. The staining time is changed for comparison of the staining effect. The staining results are shown in the following table and Figs. 1-6 .

[0057]

[0058]

[0059] The results show that, after using the methods of Examples 1-3 of the present application, the staining results are not affected within the staining time of 30-60 min, the cell staining is good, the green fluorescence is clearly visible, and the staining background is clear and without clutter. The staining results of the control group are unstable, the cell staining is too light within 30-60 min, the green fluorescence is weak, and the staining background is cluttered.

Claims

1. A visualization detection technology for autophagy activity in cotton callus cells, characterized in that, Includes the following steps: (1) Take callus samples from the same period, wash them, then add pretreatment buffer to immerse the samples and incubate to induce autophagy. (2) Discard the pretreatment buffer, add PBS buffer, and transition; (3) Discard the PBS buffer, add MDC staining solution to immerse the sample, and stain; (4) Discard the MDC staining solution and wash; Add PBS buffer, pipette and disperse to obtain callus cell suspension; (5) Prepare temporary slides of callus cells, observe green fluorescence using a fluorescence microscope, and analyze the autophagy activity of callus cells. The pretreatment buffer contains the following components at the following concentrations: 234.48 mM NaCl, 10.66 mM KCl, 52.38 mM NaHCO3, 2.02 mM NaH2PO4, and 11.12 mM D-Glucose.

2. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, The preparation method for each liter of PBS buffer is as follows: Dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 in 800mL of distilled water, adjust the pH value to 7.4 with HCl solution, and finally add distilled water to make up to 1L.

3. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, The method for preparing MDC staining solution is as follows: Take an appropriate amount of MDC stock solution (1000X) and dilute it with PBS buffer at a volume ratio of 1:1000 to obtain MDC staining solution (1X).

4. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, In step (1), the incubation temperature is 37℃ and the time is 30 to 60 minutes.

5. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, In step (2), the transition temperature is 37°C and the time is 10 min.

6. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, In step (3), the staining conditions are 37℃ in the dark for 30-60 minutes.

7. The visualization detection technology for autophagy activity of cotton callus cells according to claim 1, characterized in that, The washing reagent used was PBS buffer.

Images