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Circulating tumor cell dyeing kit and use thereof

A technology for dyeing reagents and tumor cells, which is applied in the fields of biotechnology and medicine, can solve problems such as complex procedures and long dyeing and identification operation cycles, and achieve the effects of simple preparation, true and reliable results, and improved work efficiency

Active Publication Date: 2015-05-27
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the embodiments of the present invention is to provide a circulating tumor cell staining kit and its application, aiming to solve the problems of long cycle and complicated procedures for staining and identification of circulating tumor cells

Method used

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  • Circulating tumor cell dyeing kit and use thereof
  • Circulating tumor cell dyeing kit and use thereof
  • Circulating tumor cell dyeing kit and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Preparation of circulating tumor cell staining kit:

[0047] 1. Preparation of basophilic staining solution:

[0048] Weigh 1000ml of analytical grade methanol with a graduated cylinder and add it to the beaker, weigh 1g of eosin Y with a balance, add it to the beaker, shake to fully dissolve the eosin Y, filter through a 0.22μm filter membrane, and store at room temperature for later use.

[0049] 2. Preparation of eosinophilic staining solution:

[0050] First weigh 10g of disodium hydrogen phosphate and 12.5g of potassium dihydrogen phosphate with a balance, dissolve them in 1000 ml of distilled water to make a PBS solution, then weigh 1g of medical melanin and 1g of azure I and dissolve them in the PBS solution. The pH was adjusted to 6.6, filtered through a 0.22 μm pore size filter, and stored at room temperature for later use.

[0051] 3. Sodium chloride solution preparation:

[0052] Weigh 1000ml of distilled water with a measuring cylinder and add it to the b...

Embodiment 2

[0058] Tumor cell line staining:

[0059] 1. Take 2ml of freshly passaged prostate cancer cell line PC3 and adjust the cell concentration to about 0.5×10 6 / ml, mix well.

[0060] 2. Use a pipette gun to take 1 μL (about 500 cells) of cells and add them to 25 ml of the fixative prepared in Example 1, and fix them for ten minutes.

[0061] 3. Wet the filter membrane of the filter with the sodium chloride solution prepared in Example 1.

[0062] 4. Transfer the above-mentioned fixative solution containing cells to the filter with a Pasteur tube, use negative pressure to make the fixative solution flow out through the filter membrane, and the cells are trapped on the filter membrane.

[0063] 5. Take a six-hole plate, place a piece of parafilm of appropriate size in one of the holes, and place the filter on the parafilm to prevent the liquid from flowing out of the filter.

[0064] 6. Add 300 μL of basophilic dye solution to the filter membrane, stain for 30 seconds, use negat...

Embodiment 3

[0070] Filter staining of blood samples from breast cancer patients:

[0071] 1. Use EDTA-2K anticoagulated blood collection tubes to collect 2.5ml of blood from breast cancer patients in clinical medical units.

[0072] 2. Centrifuge the blood, centrifugation conditions: 400G, 10 minutes; carefully suck off the supernatant with a pipette gun.

[0073] 3. Transfer the above blood to 25ml fixative with a pipette gun, and fix it for 10 minutes.

[0074] 4. Wet the membrane in the filter with 5% (w / v) glucose solution.

[0075] 5. Transfer the blood-containing fixative to the filter with a Pasteur tube, and use negative pressure to make it flow out through the filter. Larger cells in the blood and circulating tumor cells are trapped on the membrane.

[0076] 6. Add an appropriate amount of 5% glucose solution to rinse, use negative pressure to make it flow out, and try to remove it as clean as possible.

[0077] 7. Take a six-hole plate, put a piece of sealing film of appropri...

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Abstract

The invention provides a circulating tumor cell dyeing kit and a use thereof. The circulating tumor cell dyeing kit is suitable for the field of biotechnology and the medical science. The circulating tumor cell dyeing kit comprises a filter, a basophilic dyeing liquid, an eosinophilic dyeing liquid, an isotonic solution and a stationary liquid, wherein the basophilic dyeing liquid, the eosinophilic dyeing liquid, the isotonic solution and the stationary liquid are sub-packaged. The filter comprises a filter membrane having aperture sizes of 8 micrometers. The basophilic dyeing liquid is an eosin Y methanol solution. The eosinophilic dyeing liquid is a phosphate buffer solution containing methlene blue and an azure I. The stationary liquid is a phosphate buffer solution containing paraformaldehyde and bovine serum albumin. The circulating tumor cell dyeing kit has good dying effects, a low cost and short operation time, can be prepared simply, is convenient for operation and greatly improves work efficiency.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, in particular to a circulating tumor cell staining kit and its application. Background technique [0002] Circulating tumor cells (CTCs) are tumor cells that spontaneously shed from solid tumors or metastases or enter the peripheral blood circulation due to medical procedures. They can reside in blood, bone marrow, lymph nodes or other healthy organs and form new tumor cells. metastases. By detecting and identifying CTCs, it is helpful to find early micrometastasis, evaluate prognosis, evaluate treatment effect and realize individualized drug treatment. CTCs have become a new research hotspot in tumor biology. [0003] The current methods for detecting CTCs are mainly based on PCR or immunology, neither of which can obtain detailed cell morphology information, and the required reagents or instruments are expensive. Some people also use staining methods to identify CTCs from a morphol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/30
Inventor 李胜高杰王俊蔡从利周鹏飞
Owner WUHAN YZY MEDICAL SCI & TECH
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