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Lymphocyte separation liquid and method of separating spleen lymphocyte

A technology of lymphocytes and separation liquid, which is applied in the field of cell separation liquid and cell separation, can solve the problems of cell toxicity, cumbersome steps, poor cell viability, etc., achieve the effect of solving toxicity problems, simplifying the separation process, and improving the success rate

Active Publication Date: 2010-09-15
SHENZHEN DAKEWE BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since dextran will affect the surface protein activity of lymphocytes, and the separation solution contains impermeable ion diatrizoate (diatrizoate), it will affect the Gibbs-Donnan equilibrium of the ion-permeable membrane
Therefore, the traditional lymphocyte separation fluid is toxic to the cells, and the cell state is not good.
[0006] Second, the steps are cumbersome, highly skilled, and require high experience for the experimenter
[0008] Third, dead cells and cell debris cannot be effectively removed, resulting in poor cell viability
The final result is that the whole centrifuge tube has no clear liquid interface and no clear lymphocyte layer

Method used

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  • Lymphocyte separation liquid and method of separating spleen lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A lymphocyte separation liquid, which consists of:

[0044] Iodixanol 14% (w / v)

[0045] RPMI1640 medium 76.7% (v / v)

[0046] Ultrapure water 9.3% (v / v)

[0047] The separator:

[0048] Density 1.077g / mL (20°C);

[0049] Osmotic pressure 230mOsm;

[0050] Endotoxin <1EU / mL;

[0051] pH value 7.0-7.2(20℃)

[0052] The mouse spleen lymphocytes were isolated using this lymphocyte separation solution.

[0053] experimental method:

[0054] Mice were killed by neck dislocation and immersed in 75% ethanol for 1-2 minutes. Carefully cut off the abdominal skin of the mouse in an ultra-clean bench, fix it with pins, then cut the abdominal cavity of the mouse, and take out the mouse spleen with tweezers. Pay attention to aseptic operation.

[0055] The mouse spleen was directly ground in 3-5 mL of the above lymphocyte separation medium to prepare a single cell suspension.

[0056] Transfer the separation liquid containing the spleen cells to a centrifuge tube. Overlay...

Embodiment 2

[0060] A lymphocyte separation liquid, which consists of:

[0061] Iodixanol 15.5% (w / v)

[0062] RPMI1640 medium 74.2% (v / v)

[0063] Ultrapure water 10.3% (v / v)

[0064] The separator:

[0065] Density 1.084g / mL (20°C);

[0066] Osmotic pressure 270mOsm;

[0067] Endotoxin <1EU / mL;

[0068] pH value 7.0-7.2(20℃)

[0069] Using the lymphocyte separation liquid, the same experimental method as in Example 1 was used to separate mouse spleen lymphocytes.

Embodiment 3

[0071] A lymphocyte separation liquid, which consists of:

[0072] Iodixanol 17.5% (w / v)

[0073] RPMI1640 medium 70.8% (v / v)

[0074] Ultrapure water 11.7% (v / v)

[0075] The separator:

[0076] Density 1.095g / mL (20°C);

[0077] Osmotic pressure 310mOsm;

[0078] Endotoxin <1EU / mL;

[0079] pH value 7.0-7.2(20℃)

[0080] Using the lymphocyte separation liquid, the same experimental method as in Example 1 was used to separate mouse spleen lymphocytes.

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Abstract

The invention discloses a separating liquid of lymphocyte with Iodixanol density at 14.0-17.5%, which comprises the following steps: grinding the separated liquid of spleen; making single-cell suspension; centrifuging.

Description

technical field [0001] The invention relates to a cell separation liquid and a cell separation method, in particular to a lymphocyte separation liquid and a method for separating spleen lymphocytes. Background technique [0002] Mice and rats are the most commonly used experimental animals in biological and medical research. The spleen is the main source of lymphocytes for such animals. If the research involves the field of immunology, it is usually necessary to isolate spleen lymphocytes from mice or rats. Spleen lymphocytes are usually obtained by density gradient centrifugation. For a long time, the traditional spleen lymphocyte separation adopts a three-step method, namely: grinding, cell suspension and centrifugation. First grind the mouse spleen in cell culture medium or other physiological buffer (hereinafter, for the convenience of description, collectively referred to as culture medium) to disperse it into a single cell suspension; add common mouse spleen lymphocy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078
Inventor 朱义鑫
Owner SHENZHEN DAKEWE BIOENG
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