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Vector for assaying in-vitro cell proliferation and dynamic in-vitro cell proliferation assay method

A technology of cell proliferation and dynamic detection, applied in the field of cell detection, can solve the problems of not being able to obtain the true state of living cells, not being able to detect and analyze cells, and destroying cells, so as to reduce inactivated cells and cell fragments, with small errors and repeatability Good results

Active Publication Date: 2018-04-17
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In short, the main disadvantages of the commonly used techniques for detecting cell proliferation are: the need to label and destroy cells, so it is impossible to obtain the real state of living cells during growth; the end point method is used, which can only give the final result, so the cell status cannot be determined. Growth process as dynamic detection and analysis

Method used

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  • Vector for assaying in-vitro cell proliferation and dynamic in-vitro cell proliferation assay method
  • Vector for assaying in-vitro cell proliferation and dynamic in-vitro cell proliferation assay method
  • Vector for assaying in-vitro cell proliferation and dynamic in-vitro cell proliferation assay method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0118] Example 2: Commonly used cell proliferation detection method--MTT method

[0119] 1. Collect the logarithmic phase cells and adjust the concentration of the cell suspension to 1X 10 4 . The diluted cell suspension was added to a 96-well plate, and 200 μl was added to each well. Each sample was repeated 3-5 times, and a blank control well with medium only was set, and the edge wells were filled with sterile PBS. A total of seven 96-well plates were inoculated.

[0120] 2. Put it in a CO2 incubator, 5% CO2, and incubate at 37°C. Thereafter, the medium was changed every 2 days.

[0121] 3. On the second day, take out a 96-well plate, add 20 μl of MTT solution (5 mg / ml, ie 0.5% MTT) to each well, and continue culturing for 4 hours.

[0122] 4. Carefully aspirate and discard the supernatant in the wells, add 150 μl DMSO to each well, shake on a shaker at low speed for 10 minutes, and the crystals are fully dissolved.

[0123] 5. On the ELISA instrument, select 490nm to...

Embodiment 3

[0126] Example 3: Commonly used cell proliferation detection kit--CCK-8 method

[0127] 1. Collect logarithmic phase cells and adjust the concentration of cell suspension to 1X10 4 . The diluted cell suspension was added to a 96-well plate, and 200 μl was added to each well. Each sample was repeated 3-5 times, and a blank control well with medium only was set, and the edge wells were filled with sterile PBS. A total of seven 96-well plates were inoculated.

[0128] 2. Put it in a CO2 incubator, 5% CO2, and incubate at 37°C. Thereafter, the medium was changed every 2 days.

[0129] 3. On the second day, take out a 96-well plate, add 20 μl Cell Counting kit solution to each well, and continue to incubate for 4 hours.

[0130] 4. On the ELISA instrument, select 450nm to measure the absorbance value of each well, and calculate the average value.

[0131] 5. After every 24 hours, take out a 96-well plate and repeat steps 3-4. 6. Draw the cell growth curve with time as the X a...

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Abstract

The invention relates to a vector for assaying in-vitro cell proliferation and a dynamic in-vitro cell proliferation assay method. The vector for dynamically assaying in-vitro cell proliferation is characterized in that the vector consists of a PLKO.1 sequence, a CMV sequence, an eGFP sequence, an IRES sequence and a Puro sequence which are sequentially connected. Compared with the similar cell lines, a stably transfected cell line constructed by the invention has the advantages of high fluorescent brightness, good photostability and low background signals, and does not have significant influence on the physiology of cells. The assay method comprises the following steps: three vectors, i.e. the vector disclosed by the invention, the psPAX2 vector and the PMD2.G vector, are used for packaging slow virus; the slow virus obtained in step (1) is used for transfecting target cells, and puromycin screening and flow cytometry sorting are carried out, so that the stably transfected cell line is obtained; an appropriate number of cells are chosen to be added into a cell culture plate, and after the cells are attached to the wall, an image is acquired by a high-content cell imaging system; data are processed, and a cell proliferation curve is drawn. The vector and the method disclosed by the invention have the advantages of high automation degree, little error, good repeatability and high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of cell detection and relates to a method for dynamically detecting cell proliferation in vitro. Background technique [0002] Cell proliferation is an important feature of living organisms, which refers to the division process of cells through reactions such as DNA replication, RNA transcription, and protein synthesis under the action of cycle regulatory factors. Organisms produce new individuals through cell proliferation. When cell proliferation is out of control, it will lead to various diseases. For example, cancer is a malignant tumor formed due to the disordered proliferation of tumor cells in the body. Therefore, cell proliferation has always been one of the hotspots in medical research. Cell proliferation detection technology is widely used in research fields such as molecular biology, genetics, tumor biology, immunology, pharmacology and pharmacokinetics. As an experimental technique, it is not on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/867C12N15/66C12Q1/02
CPCC12N15/65C12N15/86C12N2740/15043G01N33/5005
Inventor 聂勇战窦建华唐光波杨美超张慧霞
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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