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Method for determining proportion of number of A cells to number of B cells in pancreatic islets

A technology of islet cells and A cells, applied in the biological field, can solve problems such as cell damage, cell death, living cell function, detection and testing, etc.

Active Publication Date: 2015-04-08
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the disadvantage of this method is that it causes damage to the cells: the islet cells are fixed before the immune response, changing their cell morphology, making the cells dead and making it impossible to perform other live cell function tests, such as probing the secretory function of the cells and many more

Method used

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  • Method for determining proportion of number of A cells to number of B cells in pancreatic islets
  • Method for determining proportion of number of A cells to number of B cells in pancreatic islets
  • Method for determining proportion of number of A cells to number of B cells in pancreatic islets

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Embodiment 1

[0036] Embodiment 1 of the present invention provides a method for determining the ratio of A cells to B cells in pancreatic islets, comprising the following steps:

[0037] Step 101: taking a part of the islet cell mass to be tested, and isolating a plurality of A cells and a plurality of B cells therefrom;

[0038] Step 102: Count the cell number m of the A cells and the cell number n of the B cells respectively; measure the total area S of the m A cells 1 and the total area S of n B cells 2 ;

[0039] Step 103: adding a zinc ion fluorescent dye to the islet culture medium, then placing the m A cells in the culture medium, and detecting the average fluorescence value a of the m A cells before fluorescence quenching after culture 1 ; Add non-fluorescein zinc ion complexing agent to the islet culture medium, and detect the average fluorescence value b after the fluorescence quenching of the m A cells after standing 1 ;

[0040] Step 104: Add zinc ion fluorescent dye to the...

Embodiment 2

[0067] Embodiment 2 of the present invention provides a preferred method for determining the ratio of the number of A cells to B cells in pancreatic islets, comprising the following steps:

[0068] Step 1: Take a part of the islet cell mass to be tested, and separate multiple A cells and multiple B cells from it;

[0069] Second step: respectively counting the cell number m of the A cells and the cell number n of the B cells; respectively measuring the total area S of the plurality of A cells 1 and the total area S of the plurality of B cells 2 ;

[0070] Step 3: Add ZRL-18-12 μmol / L to the islet culture medium, then place the m A cells in the culture medium, and culture them for 0.8-1.2 hours; then detect the fluorescence quenching of the m A cells mean fluorescence value before a 1 ; Add TPEN0.8-1.2mmol / L to the islet culture medium, let it stand for 12-25min; then detect the average fluorescence value b after the fluorescence quenching of the m A cells 1 ;

[0071] Ste...

Embodiment 3

[0080] Embodiment 3 of the present invention provides another preferred method for determining the ratio of A cells to B cells in pancreatic islets, comprising the following steps:

[0081] Step 201: taking a part of the islet cell mass to be tested, and isolating a plurality of A cells and a plurality of B cells therefrom;

[0082] Step 202: Count the cell number m of the A cells and the cell number n of the B cells respectively; measure the total area S of the m A cells 1 and the total area S of n B cells 2 ;

[0083] Step 203: Add ZRL-110 μmol / L to the islet culture medium, then place the m A cells in the medium, and culture them for 1 hour; then detect the average fluorescence value a of the m A cells before fluorescence quenching 1 ; Add TPEN 1mmol / L to the islet medium, let it stand for 20min; then detect the average fluorescence value b of the m A cells after fluorescence quenching 1 ;

[0084] Step 204: Add ZRL-110 μmol / L to the islet medium, then place the n B cel...

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Abstract

The invention relates to the technical field of biology and specifically relates to a method for determining the proportion of the number of A cells to the number of B cells in pancreatic islets. The method comprises the following steps of: taking one part out of a cell mass of the pancreatic islets, which needs to be determined, and further separating a plurality of the A cells and the B cells from the cell mass; respectively performing statistics and measuring on the number of the cells and the total area; adding a zinc ion fluorescent dye into a culture medium for the pancreatic islets, and further placing the A cells in the culture medium for culture; then detecting the average fluorescence value before fluorescence quenching of the A cells; further adding a non-fluorescence zinc ion complexing agent, standing and then detecting the average fluorescence value after fluoresence quenching of the A cells; further detecting the average fluorescence value before fluoresence quenching and the average fluorescence value after fluoresence quenching of the B cells by using the same method; further taking one part out of the cell mass of the pancreatic islets, which needs to be determined, as a detection sample, and detecting the fluoresence value before fluoresence quenching and the fluoresence value after fluoresence quenching by using the same method; and obtaining the results according to a formula in the specification. The method disclosed by the invention can be performed in living cells, and the non-destructive detection purpose can be further realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for measuring the ratio of A cells to B cells in pancreatic islets. Background technique [0002] Pancreatic islets contain various cells such as A cells, B cells, D cells, and PP cells. Among them, the ratio of A cells to B cells plays an important role in the regulation of human glucose metabolism. [0003] In the related art, the method for determining the ratio of the number of A cells to B cells in the islets is usually as follows: First, the islet cell mass is fixed with paraformaldehyde (PFA), embedded and sliced, and then the primary antibody for insulin and glucagon are added. Finally, add different secondary antibodies matching the above two antibodies (labeled with different fluorescent substances), and finally calculate the ratio of A cells to B cells by measuring the area of ​​the fluorescent part. [0004] However, the disadvantage of this method is that it c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 陈良怡王意
Owner PEKING UNIV