Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect

A technology of immune effector cells and hematoxylin, which is applied in the field of application of hematoxylin in the detection of cytotoxic effects mediated by immune effector cells, and can solve problems such as environmental pollution, high natural release rate, and specificity of FACS method

Inactive Publication Date: 2012-09-05
INST OF BASIC MEDICINE OF SAMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these two methods are sensitive and specific, the former has problems such as short half-life and high natural release rate, while the latter requires complex instruments and equipment. At the same time, both of them can cause environmental pollution, which limits their application.
Non-isotope methods include flow cytometry (FACS) method, lactate dehydrogenase method, trace NAG release method, crystal violet staining method and clone formation method, etc. Many methods are not satisfactory, such as FACS method is specific and sensitive , but requires large-scale equipment and supporting reagents, which is not suitable for grass-roots promotion and application
At present, there is no relevant research report on the use of hematoxylin staining method to measure the effect of cell-mediated cytotoxicity

Method used

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  • Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect
  • Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect
  • Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Detection of Hep2 cell proliferation activity

[0025] (1) Detecting the proliferation activity of Hep2 cells by hematoxylin staining: add RPMI1640 medium (complete medium) containing 100 ml / L calf serum into a 96-well cell culture plate, 100 μl / well. Hep2 cells were digested with 2.5 g / L trypsin, the supernatant was discarded after centrifugation, and the corresponding cell concentration was adjusted with complete medium. Then, 100 μl of the cell suspension was added to each corresponding well of the above 96-well cell culture plate. After mixing, it was diluted by a factor of 2, and 8 dilutions were set up in sequence, each dilution was 3 duplicate wells, and then each well was supplemented with complete medium. to 200 μl, and set the microplate reader to zero well. Set the 96-well cell culture plate at 37°C, 50ml / L CO 2 Incubate in a constant temperature saturated humidity incubator for 44 hours. Take out the 96-well cell culture plate and discard the s...

Embodiment 2

[0028] Example 2: Detection of cytotoxic effects of human NK cell lines:

[0029] (1) Hematoxylin staining method to detect the cytotoxic effect of human NK cell lines:

[0030] I. Culture expansion of human NK cell line: NK92 cell culture medium containing 125ml / L fetal bovine serum, 125ml / L horse serum and 1×10 5 U / L rhIL-2 in α-MEM medium, passaged for 2-3 days. NKL cell culture medium containing 100ml / L fetal bovine serum and 1×10 5 U / L rhIL-2 in RPMI1640 medium, passaged for 2-3 days. YT cell culture medium is RPMI1640 medium containing 100 ml / L calf serum, and passaged for 2-3 days.

[0031] II. Detection of cytotoxic effect of human NK cell line: 2.5ml / L trypsin digested Hep2 and SGC7901 cells, and after centrifugation, the number of cells was adjusted to 1×10 with complete medium 8 / L, added to 96-well cell culture plate, set at 37°C, 50ml / L CO 2Incubate in a constant temperature saturated humidity incubator for 12 hours to make it adherent. NK92, NKL and YT cell...

Embodiment 3

[0040] Example 3: Detection of cytotoxic effect of rabbit CTL cells

[0041] (1) Hematoxylin staining method to detect the cytotoxic effect of rabbit CTL cells: Digest VX2 cells with 2.5ml / L trypsin, adjust the corresponding cell concentration with complete medium after centrifugation and washing, and add it to each corresponding well of a 96-well cell culture plate , 100μl / well; then place the 96-well cell culture plate at 37°C, 50ml / L CO 2 Incubate in a constant temperature saturated humidity incubator to make it adherent. New Zealand white rabbits bearing tumors (VX2 cells) for 12 days were sacrificed, and the spleen was aseptically removed, cut into pieces, and ground with a 100-mesh steel mesh to prepare a single cell suspension of the spleen. In each corresponding well of the culture plate, 100 μl / well. Four effector-to-target ratios (E:T) of 40:1, 20:1, 10:1 and 5:1 were set, and the corresponding CTL effector cell control and VX2 target cell control were set, all of ...

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Abstract

The invention discloses a method for measuring a cellulotoxic effect of a cell-mediated immunity effect by using hematoxylin. The method comprises the following steps of placing adherent tumor cell suspension in a cell culture plate; adding immune effector cell to be detected after adherence to form a certain effector-target ratio; taking the mixture out and discarding supernate after being carried out co-incubation for a proper time in a culture tank; adding 4% of paraformaldehyde immediately into the mixture and fixing for 30m after removing effector cell by washing the plate for third times by using PBS; adding hematoxylin staining solution 100 mu ml per hole; washing with distilled water for three to five times to remove uncombined dye after staining for 20m, and airing in room temperature; adding 1% of hydrochloric acid alcohol for decoloration 100 mu ml per hole; measuring the absorbance value at the position of 540nm by a microplate reader immediately after decoloration and recording the absorbance value; calculating the kill rate for each effector target ratio according to the formula shown in the Figure Researches show that the invention is specific, stable, simple, economical and the like.

Description

technical field [0001] The present invention relates to the application of hematoxylin in detecting the cytotoxic effect mediated by immune effector cells. Background technique [0002] Immune effector cells such as NK, CTL and DC have important antitumor effects. With the in-depth progress of tumor immunology research, the method for measuring the cell-mediated cytotoxic effect has emerged, which has laid the foundation for the basic and clinical research of tumor biotherapy. At present, there are many methods for detecting cell-mediated cytotoxic effects, which can be basically divided into two categories: isotopic methods and non-isotopic methods. Isotope method including 4h 51 Cr release method and 3 H-TdR incorporation method. Although these two methods are sensitive and specific, the former has the problems of short half-life and high natural release rate, while the latter requires complex instruments and equipment, and both can cause environmental pollution and li...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王郡甫张建华陈红
Owner INST OF BASIC MEDICINE OF SAMS
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