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Carrier for detecting in vitro cell proliferation and in vitro cell proliferation dynamic detection 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, etc., to achieve the effect of small error, less human participation, and good repeatability

Active Publication Date: 2021-09-28
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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  • Carrier for detecting in vitro cell proliferation and in vitro cell proliferation dynamic detection method
  • Carrier for detecting in vitro cell proliferation and in vitro cell proliferation dynamic detection method
  • Carrier for detecting in vitro cell proliferation and in vitro cell proliferation dynamic detection method

Examples

Experimental program
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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 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 axis an...

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Abstract

The invention relates to a carrier for detecting in vitro cell proliferation and a dynamic detection method for in vitro cell proliferation. The carrier for dynamic detection of in vitro cell proliferation is characterized in that the carrier includes: sequentially connected PLKO.1 sequence, CMV sequence, eGFP sequence, IRES sequence and Puro sequence. Compared with similar cell lines, the stably transfected cell lines constructed by the present invention have strong fluorescence brightness, good photostability, low background signal, and no significant influence on cell physiology. The detection method includes: using the carrier of the present invention, the psPAX2 carrier and the PMD2.G carrier three vectors to package the lentivirus; using the lentivirus obtained in step 1 to transfect target cells, puromycin screening, and flow cytometry sorting to obtain Stably transform the cell line; select the appropriate number of cells and add them to the cell culture plate. After the cells adhere to the wall, the high-content cell imaging system collects images; processes the data and draws the cell proliferation curve. The invention has high degree of automation, small 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 Patents(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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