A method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model

Abstract

The present invention discloses a method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model, comprising the following steps: Fix a small round piece with a diameter of 14 mm on a square glass slide using a dental orthodontic rubber band. After autoclaving, inoculate cells onto the square glass slide according to the original method, place it on the rotating chamber fixing rack, and conduct a rotating simulated weightlessness experiment; after rotation is completed, take out the square glass slide, remove the small round piece and conduct a cell immunofluorescence experiment. When incubating the primary antibody, stick a plastic wrap on the back of the lid of a 24-well plate, then drop 30 μL of the primary antibody on the lid, and invert the side of the small round piece with cells onto the droplet; the same method is used for secondary antibody incubation. The present invention can minimize the usage amount of the primary antibody and the secondary antibody during immunofluorescence without affecting the immunofluorescence effect and without the need for an immunohistochemical pen, and the samples can be fully utilized.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a method for cell immunofluorescence operation under a 2D rotary cell simulated weightlessness model. Background Art

[0002] A 2D rotator is a gravity research platform that simulates the weightlessness effect of cells through ground equipment. It can make the sample rotate around an axis perpendicular to the gravity field, and its rotation speed must be fast enough to reach a state where the rotating system no longer perceives the gravity vector of rapid rotation, so as to achieve the purpose of simulating the weightlessness effect. A large number of studies have found that the experimental results under simulated microgravity conditions are basically the same as or similar to those under real microgravity conditions, which also supports that this rotator experimental platform can be used as an effective tool for simulating the microgravity effect of cells.

[0003] The morphology and molecular localization of cells after being rotated to simulate the weightlessness effect are one of the important methods for studying space biology. At present, the 2D rotary simulation of weightlessness basically adopts the process of inoculating cells on a square glass slide of 2.55×2.15 cm, taking out the glass slide from the rotary cabin after rotary simulation of weightlessness, and then scraping the cells off the glass slide for subsequent operations such as protein extraction or RNA extraction detection. If immunofluorescence is to be performed, the glass slide needs to be placed in a 6-well plate, and the primary antibody and secondary antibody are added according to the steps of the immunofluorescence experiment, and finally observed with a fluorescence microscope. However, this technique has two obvious disadvantages when applied to cells in the rotary simulated weightlessness model. One is the waste of antibodies. When a glass slide covered with cells is placed in a 6-well plate, at least 100 μL of the primary antibody and secondary antibody dilution solution need to be added. Since antibodies, especially primary antibodies, are expensive, operating in this way will increase the operating cost. If an immunostaining pen is used to draw a circle on the glass slide to enclose the antibodies, a part of the antibodies can be saved, but the circle drawn by the immunostaining pen is easy to fall off, and once a little falls off, the antibody dilution solution will flow out, resulting in all previous efforts being wasted. The other is the waste of cells. Performing immunofluorescence on a square glass slide will waste all the cells on the entire glass slide. The fluorescence microscope often does not need to observe so many cells, and the cells around the glass slide will be wasted. The operation of constructing the rotary simulated weightlessness model is very complicated and requires a large amount of culture medium. Some cells are also relatively precious, so only performing the immunofluorescence experiment after rotation is somewhat wasteful. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for cell immunofluorescence operation under a 2D rotary cell simulated weightlessness model, which can minimize the usage amount of the primary antibody and secondary antibody when performing immunofluorescence without affecting the immunofluorescence effect and without the need for an immunostaining pen. In addition, the cells on the square glass slide except for the small round pieces can continue to be used for immunoblotting or PCR and other detections, saving precious samples.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model, comprising the following steps:

[0007] S1. Fix a small round piece with a diameter of 14 mm on a square glass slide using a dental orthodontic rubber band. After high-pressure steam sterilization, inoculate cells onto the square glass slide according to the original method, place it on the rotating cabin fixing frame, and conduct a rotating simulated weightlessness experiment;

[0008] S2. After rotation is completed, take out the square glass slide, remove the small round piece and conduct a cell immunofluorescence experiment. When incubating the primary antibody, stick the plastic wrap on the back of the lid of the 24-well plate, then drop 30 μL of the primary antibody on the lid, and invert the side of the small round piece with cells on the droplet; the same method is used for secondary antibody incubation.

[0009] Further, when washing the primary antibody or secondary antibody, place the small round piece in the 24-well plate for washing.

[0010] Further, the small round piece with a diameter of 14 mm is fixed at the central position of the square glass slide, and the dental orthodontic rubber band is sleeved outside the small round piece and the square glass slide.

[0011] When using the method of the present invention for immunofluorescence, there is no difference in the effect compared with the traditional method. The method of the present invention has the following beneficial effects:

[0012] 1) It can greatly reduce the usage amount of the primary antibody and secondary antibody during the cell immunofluorescence operation process. Only about 30 μL of the primary antibody dilution and secondary antibody dilution are needed for each small round piece.

[0013] 2) Except for the cells on the small round piece for immunofluorescence, other cells on the square glass slide can continue to extract proteins, RNA or conduct other experiments, which avoids waste of samples for relatively precious cell samples and makes full use of the samples. Description of the Drawings

[0014] By reading the detailed description of the non-restrictive embodiments with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

[0015] Figure 1 Schematic diagram of the structure for fixing the small round piece on the square glass slide.

[0016] Figure 2 Schematic diagram of the staining of Hsp60 molecules in primary human umbilical vein endothelial cells using the method of the present invention and the traditional method in the embodiment of the present invention. Detailed Embodiments

[0017] The present invention will be described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all fall within the protection scope of the present invention.

[0018] Example 1

[0019] A method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model, comprising the following steps:

[0020] S1. As Figure 1 shown, use a dental orthodontic rubber band 3 to fix a small round piece 1 with a diameter of 14 mm on a square glass slide 2. After high-pressure steam sterilization, inoculate cells onto the square glass slide according to the original method, place it on the rotating cabin fixing frame, and conduct a rotating simulated weightlessness experiment; specifically, the small round piece with a diameter of 14 mm is fixed at the center position of the square glass slide, and the dental orthodontic rubber band is sleeved outside the small round piece and the square glass slide. After high-pressure steam sterilization, inoculate cells onto the square glass slide at a density of 1×10 5 cells / slice, place it in a six-well plate and culture overnight. Fill the rotating cabin with ECM complete medium and preheat it for 24 h. After the cells grow to about 50% on the square glass slide, insert the glass slide with cells into the card slot of the stainless steel bracket in the rotating cabin, exhaust the air bubbles, and then place it on the rotating arm of the rotator and rotate and culture it in a 37°C incubator at a speed of 24 r / min.

[0021] S2. After rotation is completed, take out the square glass slide, remove the small round piece and conduct a cell immunofluorescence experiment. When incubating the primary antibody, stick the plastic wrap on the back of the lid of the 24-well plate, then drop 30 μL of the primary antibody on the lid, and invert the side of the small round piece with cells onto the liquid drop; the same method is used for incubating the secondary antibody. Among them, when washing the primary antibody or the secondary antibody, place the small round piece in the 24-well plate for washing.

[0022] As Figure 2 shown, using this method and the traditional method to stain the Hsp60 molecule of primary human umbilical vein endothelial cells, there is no difference in the staining effect.

[0023] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence of the present invention.

Claims

1. A method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model, characterized in that: The steps are as follows: S1. Fix a small round piece with a diameter of 14 mm on a square glass slide using a dental orthodontic rubber band. After high-pressure steam sterilization, inoculate cells onto the square glass slide according to the original method, place it on the rotary cabin fixing rack, and conduct a rotary simulated weightlessness experiment; S2. After the rotation is completed, take out the square glass slide, remove the small round piece and conduct a cell immunofluorescence experiment. When incubating the primary antibody, stick the plastic wrap on the back of the lid of the 24-well plate, then drop 30 μL of the primary antibody on the lid, and place the side of the small round piece with cells upside down on the droplet; the same method is used for the secondary antibody incubation.

2. The method for cell immunofluorescence operation under a 2D rotating cell simulated weightlessness model according to claim 1, wherein: When washing the primary antibody or the secondary antibody, place the small round piece in the 24-well plate for washing.

3. The method for cell immunofluorescence operation under a 2D rotary cell-simulated weightlessness model according to claim 1, wherein: The small round piece with a diameter of 14 mm is fixed at the center position of the square glass slide, and the dental orthodontic rubber band is sleeved outside the small round piece and the square glass slide.

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