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Method for preparing cell nucleus with controllable aggregation state and capacity of being preserved for a long time

A technology of aggregation state and long-term storage, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of long delivery cycle and high price, achieve low price, reduce application cost, and good product development potential Effect

Active Publication Date: 2017-06-20
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is currently no public development plan for nuclear calibration reagents at home and abroad. The commercially available nuclear reagents for flow cytometer calibration are mainly monopolized by BD and Biosure, which are expensive and have a long delivery cycle. a lot of inconvenience

Method used

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  • Method for preparing cell nucleus with controllable aggregation state and capacity of being preserved for a long time
  • Method for preparing cell nucleus with controllable aggregation state and capacity of being preserved for a long time
  • Method for preparing cell nucleus with controllable aggregation state and capacity of being preserved for a long time

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Experimental program
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Effect test

Embodiment approach 1

[0041] (1) Collect 3*10 7 The chicken erythrocytes were washed with PBS, centrifuged at 1500rpm to remove the supernatant, and 10 mL of lysate was added, which contained 0.1% Triton X-100, 1 μM dithiothreitol and 0.5 μM phenylmethylsulfonyl fluoride, 1 % ethyl phenyl polyethylene glycol, 40Mm sodium citrate, 0.05% DMSO, pH7.6.

[0042] (2) After lysing for 10 minutes, the chicken erythrocyte solution was slowly added to the upper layer of 30 mL concentrated sucrose solution, and centrifuged at 30,000 g at 4° C. for 50 minutes. The sucrose solution contained 2M sucrose, 1% glycerol, and 0.2% glycine dissolved in 0.01M PBS buffer.

[0043] (3) Get the supernatant, and add the chicken erythrocyte nuclei obtained into -20 ℃ pre-cooled fixative solution, which contains 40% methanol, 30% ethanol, 1% glutaraldehyde, 1% formaldehyde, 1 % bovine serum albumin, 1% glycerin, and the rest are filled with water. After fixation, the cells showed different degrees of multi-aggregate struc...

Embodiment approach 2

[0046] In this case, the cells in Embodiment 1 were replaced with calf thymidine cells, and the rest remained unchanged. The obtained calf thymidine cell nuclei were stained with 5 μg / ml propidium iodide, and detected by flow cytometry, forming 7 peaks, and the ratio was in accordance with demand, the single-peak CV values ​​are between 1.8%-3.0% ( image 3 ), which can also meet the needs of subsequent calibration tests.

Embodiment approach 3

[0048] In this case, the cells in Embodiment 1 were replaced with zebrafish red blood cells, and the rest remained unchanged. The obtained zebrafish red blood cell nuclei were stained with 5 μg / ml propidium iodide, and detected by flow cytometry. Eight peaks were formed, and the ratio met the requirements. The peak CV values ​​are between 2.1% and 2.9%, which can also meet the needs of subsequent calibration tests.

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Abstract

The invention relates to a method for preparing a cell nucleus with a controllable aggregation state and capacity of being preserved for a long time. The method comprises the following steps: collecting nucleated cell samples, washing, centrifuging to remove the supernatant, and adding lysate for lysing; adding a sucrose solution, and centrifuging at 15000-40000g under the temperature of 4 DEG C; taking the supernatant, adding the obtained cell nucleus into stationary liquid 1 pre-cooled to a temperature of 20 DEG C below zero for crosslinking; and replacing the stationary liquid 1 with stationary liquid 2 within 2 days after crosslinking, thereby obtaining the cell nucleus with the controllable aggregation state and capacity of being preserved for a long time. According to the scheme, the cell nucleus preparation method can be completed by three steps only, and the scheme can serve as an excellent product development scheme; dependence of domestic flow cytometry on imported reagents is gradually changed, and the application cost is greatly reduced; and meanwhile, mass production of the reagents can push cells ploid detection and clinical tumor prognosis in scientific research, and the tumor treatment quality is improved. The red cell nucleus product obtained in the scheme can be stably preserved for one year or longer at the temperature of 4 DEG C and has excellent product development potential.

Description

technical field [0001] The invention belongs to the field of reagents for medical detection, and in particular relates to a preparation method of cell nuclei with controllable aggregation state and long-term preservation. Background technique [0002] Flow cytometry is one of the most advanced instruments in the fields of life science and clinical cell cycle and apoptosis detection, as well as tumor detection and prognosis. , apoptotic status and ploidy were analyzed in detail. During the above detection process, it is often necessary to calibrate the accuracy of the instrument. As the main storage place of genetic material in the cell, the nucleus has a stable and consistent DNA content, and after special treatment, it can form 1, 2, 3, 4, up to 7 or more nuclei aggregates, which can be stained with fluorescent dyes Finally, the linearity, detection limit and amplification gain of the flow cytometer are calibrated, and it can also be used as an internal standard in DNA de...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2565/626
Inventor 殷建尹焕才田晶晶白鹏利陈名利王钧
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI