Cell periodic detection kit

A detection kit and cell cycle technology, which is applied in measuring devices, microbial measurement/inspection, instruments, etc., can solve the problems of not being able to label living cells, many sample processing steps, and not being able to penetrate the cell membrane, so as to achieve convenient on-boarding time arrangement, DAPI pollution is small, and the effect of simple process

Inactive Publication Date: 2018-11-23
INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, PI is a macromolecular dye, which cannot penetrate the complete cell membrane and cannot label living cells. Before staining, the sample needs to be fixed and permeabilized. The sample processing steps are many and time-consuming.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Mouse fibroblasts were purchased from Suzhou Beinan Chuanglian Biotechnology Co., Ltd. No. BNCC100032. DMEM medium (Gibco), calf serum BSA (Gibco), trypsin (Gibco) / Triton X-100 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), Cystain DNA 1step (Partec), Alexa 647 rat anti-phospho-Histone H3 (pS28) (BD, BD Bioscience, catalog number: 558217), Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 48 (Thermo) where flow cytometry The instrument has a UV laser light source.

[0023] Mouse fibroblasts cryopreserved in liquid nitrogen were revived in a 38°C water bath, and inoculated in culture flasks. 2 When the cell fusion rate reached 80%, it was digested into a single cell suspension with 0.25% trypsin, and the digestion was terminated with DMEM medium containing 10% serum, the cells were collected by centrifugation, and washed with Dulbecco's phosphate buffer saline 3 times, 15 min each time, and then centrifuged to collect cells.

[00...

Embodiment 2

[0026] Mouse fibroblasts cryopreserved in liquid nitrogen were revived in a 38°C water bath, and inoculated in culture flasks. 2 When the cell fusion rate reached 80%, it was digested into a single cell suspension with 0.05% trypsin, and the digestion was terminated with DMEM medium containing 10% serum, the cells were collected by centrifugation, and washed with Dulbecco's phosphate buffer saline 3 times, 15 min each time, and then centrifuged to collect cells.

[0027] Wherein, a hypotonic lysis buffer is prepared, and the hypotonic lysis buffer includes deionized water, 0.1% sodium citrate by weight and volume, 0.5% NP-40 by volume and 1.5 mM EDTA. Add 100ul hypotonic lysis buffer and 5.0ul phosphorylated histone H to the cells collected by centrifugation 3 The primary antibody was incubated at room temperature for 2 hours in the dark, and then washed 3 times with Dulbecco's phosphate buffered saline, 15 min each time. Finally, 0.2ul of secondary antibody was added, incub...

Embodiment 3

[0029] Mouse fibroblasts cryopreserved in liquid nitrogen were revived in a 38°C water bath, and inoculated in culture flasks. 2 When the cell fusion rate reached 80%, it was digested into a single cell suspension with 0.5% trypsin, and the digestion was terminated with DMEM medium containing 10% serum, the cells were collected by centrifugation, and washed with Dulbecco's phosphate buffer saline 3 times, 15 min each time, and then centrifuged to collect cells.

[0030]Wherein, a hypotonic lysis buffer is prepared, and the hypotonic lysis buffer includes deionized water, 0.1% sodium citrate by weight and volume ratio, 0.3% NP-40 by volume ratio and 1.8mM EDTA. Add 120ul hypotonic lysis buffer and 0.5ul phosphorylated histone H to the cells collected by centrifugation 3 The primary antibody was incubated at room temperature for 2 hours in the dark, and then washed 3 times with Dulbecco's phosphate buffered saline, 15 min each time. Finally, 2.0ul of secondary antibody was add...

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PUM

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Abstract

The invention discloses a cell periodic detection kit. The cell periodic detection kit comprises a reagent I and a reagent II; the reagent I comprises a DMEM culture medium, 0.05 to 0.5 percent trypsin and cleaning buffering solution; and the reagent II comprises 2 to 5 ml of Cystain DNA1 step, 100 to 120 micro liter of low-permeability cracking buffering solution, 0.5 to 5 micro liter of phosphoric acid histochemical protein H3 primary antibody and 0.2 to 2.0 micro liter of phosphoric acid histochemical protein H3 secondary antibody. The kit utilizes flow cytometry technology, so that the cell period can be rapidly and accurately determined, and the period G2 and the period M can be distinguished.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell cycle detection kit. Background technique [0002] The cell cycle refers to the process of cells that divide continuously, starting from the completion of one division and ending when the next division is completed. The cell cycle is the basic process of cell life activities. Determining the cell cycle of cultured cells is beneficial to the study of cell growth and DNA synthesis and metabolism of cells. A cell cycle can be artificially divided into four consecutive periods, namely G 1 phase, S phase, G 2 Phase and M phase, each period in the cell cycle is also often called phase, and DNA content changes periodically with phase changes. DNA is labeled with nucleic acid dyes and analyzed by flow cytometry to obtain the distribution of cells at each stage and calculate the G 1 %, S% and G 2 / M%, to understand the proliferation ability of cells. [0003] Phosphorylation disr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N15/14
CPCG01N15/14G01N33/5005
Inventor 李德冠董银萍吴静王梅芳
Owner INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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