Cytoperm capable of being used as hemolytic agent and using method thereof

A technology of membrane breaking agent and hemolytic agent, which is applied in the field of medical testing reagents, can solve the problems of target cell loss, incomplete lysis, and high price, and achieve the effect of convenient application, less cell fragments, and good stability

Inactive Publication Date: 2010-01-06
张晖
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ubiquitous disadvantages such as single use, incomplete lysis, loss of target cells, and high price

Method used

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  • Cytoperm capable of being used as hemolytic agent and using method thereof
  • Cytoperm capable of being used as hemolytic agent and using method thereof
  • Cytoperm capable of being used as hemolytic agent and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Analytical-grade pure reagents were used to prepare membrane breaking agent and concentrated diluent solutions. First, 1 g of paraformaldehyde was accurately weighed, placed in a certain amount of 0.01M phosphate buffered saline (Phosphate buffered saline, PBS for short), and placed in a 37°C water bath. Bath in neutral water for 1 hour, add 0.01M sodium hydroxide (NaOH) dropwise to help dissolve the insoluble paraformaldehyde until completely dissolved, and cool to room temperature. Add accurately weighed saponin 0.5g, sodium azide 0.05g until completely dissolved, make up the volume with 0.01M PBS to make the weight 100g; adjust the pH to 7.2 (25°C) with 1N hydrochloric acid (HCl) ; Finally, use a G5 funnel to sterilize and filter to obtain a solution of membrane disrupting agent A.

[0053] Accurately weigh 10 g of paraformaldehyde, place in a certain amount of 0.1M phosphate buffered saline (Phosphate buffered saline, PBS for short), and bathe in a 37°C water bath f...

Embodiment 2

[0055] Example 2 The use method of immunophenotyping as a hemolytic agent in the detection of clinical blood samples by flow cytometry

[0056] Whole blood samples stored at room temperature and anticoagulated with EDTA, heparin or sodium citrate used within 24 hours are applied with the hemolytic agent of the present invention.

[0057] 1. Take 50 μl of anticoagulated blood and put it into the flow tube.

[0058] 2. Add an appropriate amount of fluorescently labeled antibody (mouse anti-human CD123-PE), mix well, and incubate at room temperature in the dark for 20 minutes.

[0059] 3. Add 1ml of the hemolytic agent of the present invention (the content of saponin is 0.05%, the mixed dilution of membrane breaking agent A liquid and diluent), shake evenly, place at room temperature for 5 minutes, centrifuge (300g, 5 minutes) to remove the supernatant liquid.

[0060] 4. Fix with 300 μl of 1% paraformaldehyde, and store in the dark at 4°C. Within 48 hours, it was detected and...

Embodiment 3

[0061] Example 3 The method of using flow cytometry as a membrane breaking agent to detect multiple immunological indicators in cells of clinical blood samples

[0062] Store at room temperature, use EDTA, heparin or sodium citrate anticoagulated whole blood samples within 24 hours, and apply the membrane breaking agent and hemolyzing agent involved in the present invention.

[0063] Proceed as follows:

[0064] 1. Take 50 μl of anticoagulated blood and put it into the flow tube.

[0065] 2. Add 20 μl of fluorescently labeled antibody (mouse anti-human CD3-PerCP) for detecting cell surface molecules, mix well, and incubate at room temperature in the dark for 20 minutes.

[0066] 3. Add 1ml of the hemolytic agent of the present invention (the content of saponin is 0.05%, the mixed dilution of membrane breaking agent A liquid and diluent), shake evenly, place at room temperature for 5 minutes, centrifuge (300g, 5 minutes), and remove the supernatant liquid.

[0067] 4. Resusp...

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PUM

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Abstract

The invention relates to a cytoperm capable of being used as a hemolytic agent, which comprises the main components of saponin, sodium azide and paraformaldehyde. Proper amount of the components are mixed, and dissolved in phosphate buffer to prepare the cytoperm; and the cytoperm can be used as the hemolytic agent after dilution. Compared with the prior hemolytic agent and cytoperm, the cytoperm related to the invention can be directly used as the cytoperm, also can be used as the hemolytic agent after the dilution, and has double purposes. The hemolytic agent and cytoperm can be applied to a flow cytometer to detect various immunological indexes in human body blood, has obviously superior detection specificity, sensitivity and the like than imported reagents, and particularly has the advantages in pretreatment processes such as hemolysis, perm and the like on whole blood specimens of patients with leukemia, globalism and severe liver diseases.

Description

technical field [0001] The invention belongs to the field of reagents for medical detection. In particular, it relates to reagents for auxiliary detection of flow cytometry. Background technique [0002] With the popularization and application of flow cytometer, the development and manufacture of its supporting reagents are becoming more and more important. At present, many domestic hospitals and laboratories use hemolytic agents and membrane-breaking agents imported from abroad, such as products from companies such as BDIS, BD-Pharmingen, Immunotech, and Caltag. There are common disadvantages such as single use, incomplete lysis, loss of target cells, and high price. Contents of the invention [0003] Aiming at the shortcomings of the prior art, the present invention provides a membrane disrupting agent for flow cytometry analysis and a diluted hemolyzing agent, which is obviously better than imported reagents in terms of detection specificity and sensitivity, and the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N1/28
Inventor 张晖王福生
Owner 张晖
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