Mesothelin detection cell quality control piece

A technology of cytoplasm and mesothelin, which is applied in the direction of biological testing, material inspection products, preparation of test samples, etc. It can solve the problems that there are no control cells with different expression intensities, and the control effect needs to be improved.

Pending Publication Date: 2019-12-03
REMEGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quality control sheets used in immunohistochemical experiments in the prior art do not have control cells with different expression intensities, so the control effect needs to be improved

Method used

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  • Mesothelin detection cell quality control piece
  • Mesothelin detection cell quality control piece
  • Mesothelin detection cell quality control piece

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Screening of Cell Quality Control Sheet Cells

[0084] 1. Flow Cytometry Screening

[0085] (1) Cell collection: the cells to be detected were digested with 0.25% trypsin and collected, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0086] (2) Fixation: add an appropriate amount of 4% paraformaldehyde to the collected cells to resuspend the cells, place at 4°C for fixation for 30 minutes; centrifuge with PBS at 2000 rpm for 3 minutes and wash 3 times, discard the supernatant.

[0087] (3) Counting: Resuspend the cells in 1ml of PBS (the PBS is phosphate buffered saline) for all cell lines, and count them with a cell counter. Centrifuge for 3 minutes and discard the supernatant.

[0088] (4) Primary antibody: Resuspend the cell lines in 200 μl PBS respectively, and add primary antibody (the primary antibody is an antibody that specifically recognizes MSLN protein) according to the experimental protocol to keep the final concentration...

Embodiment 2

[0122] Example 2: Paraffin embedding of cells and method for sectioning

[0123] 2.1 Cell paraffin embedding method

[0124] Experimental Materials:

[0125] MSLN four-in-one cell quality control sheet includes four cell lines: 293( Number: CRL-1573 TM ), CFPAC-1 ( Number: CRL-1918 ), OVCAR-3 ( Number:HTB-161 ) and OVCAR-3-MSLN cells were stably transfected with the humanized MSLN gene to form a cell line capable of stably overexpressing the MSLN protein. Materials used in the cell culture and embedding processes can be purchased commercially.

[0126] experiment procedure:

[0127] 1. Cell culture

[0128] Cell name: 293 (staining intensity is 0)

[0129] Medium: Gibco RPMI1640 medium + 10% FBS

[0130] Cell name: CFPAC-1 (staining intensity is 1)

[0131] Medium: Gibco IMDM medium + 10% FBS

[0132] Cell name: OVCAR-3 (staining intensity is 2)

[0133] Medium: Gibco RPMI1640-A10491-01 medium + 20% FBS + 0.01mg / ml insulin

[0134] Cell name: OVCAR-3-MSLN (...

Embodiment 3

[0157] Example 3: How to use cell quality control sheet

[0158] Experimental Materials

[0159] The main experimental materials include ethanol, xylene, citrate antigen retrieval solution, DAB chromogenic kit and other materials can be obtained through commercial channels.

[0160] Specific experimental steps:

[0161] (1) Baking slices: Turn on the constant temperature blast drying oven and set it to preheat at 65°C. When the temperature of the constant temperature blast drying oven rises to 65°C, take paraffin slices and put them on the slice rack, and put them in the oven to bake the slices for 1 hour.

[0162] (2) Dewaxing and rehydration: After the baking of the slices, take out the paraffin sections and quickly transfer them to xylene, and dewax them at room temperature for 20 minutes. Then rehydrate with gradient ethanol in the following order, absolute ethanol: 5 min → 95% ethanol: 5 min → 75% ethanol: 5 min, and then transfer to PBST for soaking for later use. (Th...

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Abstract

The invention relates to a mesothelin immunohistochemical detection cell quality control piece and a related detection reagent. The cell quality control piece comprises a sectioning area, wherein paraffin sections are adhered to the sectioning area, and the paraffin sections contain four kinds of cell clusters for expressing negative, weak positive, positive and strong positive properties of the mesothelin. The immunohistochemical detection cell quality control piece involved in the invention is mainly used for improving the accuracy and the consistency of immunohistochemical detection results, and has a high application prospect.

Description

technical field [0001] The invention relates to a cell or tissue sample control quality control sheet for immunohistochemical detection of mesothelin and related detection reagents. Background technique [0002] Mesothelin (MSLN) is a membrane protein. The mesothelin gene encodes a 71KD preprotein, which is modified and cut into two segments, 40KD glycosylated phosphatidylinositol cell membrane-anchored protein and 31KD The free fragment is called megakaryocyte-promoting factor. Studies have confirmed that the 40KD membrane-anchored protein contains a binding region with another tumor marker CA125 protein, which may be related to cell adhesion, so it is speculated that it may contribute to the distant metastasis of primary cancer (such as peritoneal metastasis) , and lead to poor prognosis. [0003] Although the function of Mesothelin is not yet clear, both the cell membrane binding region and the free region have been proven to be markers and therapeutic targets for vario...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N1/36G01N1/28
CPCG01N33/68G01N1/36G01N1/28
Inventor 于占娇毛海燕王庆波李壮林李元浩
Owner REMEGEN CO LTD
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