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Rat bone marrow derived mast cell culture method

A mast cell and culture method technology, applied in cell dissociation methods, cell culture active agents, animal cells, etc., can solve the problems of low mast cell purity, affecting experimental progress, long culture period, etc., achieving short maturation time, separation The effect of high purity and improved purity

Pending Publication Date: 2020-11-24
LABREAL BIOTECH KUNMING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to provide a method for culturing rat bone marrow-derived mast cells to solve the problem of the low purity of mast cells cultured by the existing rat bone marrow-derived mast cell culture method proposed in the above-mentioned background technology, and the culture period is long, which affects the experimental results. progress problem

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Rat bone marrow-derived mast cell culture method, the method comprises the following steps:

[0023] S1: Young SD rats born 3-5 weeks old were killed by neck dislocation, soaked in 75% alcohol for 3 minutes, and then transferred to an ultra-clean workbench for operation. Cut off the skin of the hind limbs, remove the leg muscles, and expose the femur. The external muscles of the femur are stripped clean, and the femur is cut off;

[0024] S2: Cut off the femoral head at both ends of the femur, use a 5ml syringe to absorb DMEM / F12 basal medium, blow out the femoral bone marrow, and rinse the bone marrow during the blowing process;

[0025] S3: Use a Pasteur pipette to blow the bone marrow 30 times to make a single-cell suspension, centrifuge through a centrifuge, collect the cells, and discard the supernatant;

[0026] S4: Add 3ml of red blood cell lysate and lyse at 4 degrees Celsius for 3 minutes, then centrifuge through a centrifuge to collect the cells, discard the ...

Embodiment 2

[0035] Rat bone marrow-derived mast cell culture method, the method comprises the following steps:

[0036] S1: Young SD rats born 3-5 weeks old were killed by neck dislocation, soaked in 75% alcohol for 3 minutes, and then transferred to an ultra-clean workbench for operation. Cut off the skin of the hind limbs, remove the leg muscles, and expose the femur. The external muscles of the femur are stripped clean, and the femur is cut off;

[0037] S2: Cut off the femoral head at both ends of the femur, use a 10ml syringe to absorb DMEM / F12 basal medium, blow out the femoral bone marrow, and rinse the bone marrow during the blowing process;

[0038] S3: Use a Pasteur pipette to blow the bone marrow 30 times to make a single-cell suspension, centrifuge through a centrifuge, collect the cells, and discard the supernatant;

[0039] S4: Add 6ml of red blood cell lysate and lyse at 4°C for 3 minutes, then centrifuge to collect the cells, discard the supernatant, then absorb 4ml of ma...

Embodiment 3

[0048] Rat bone marrow-derived mast cell culture method, the method comprises the following steps:

[0049] S1: Young SD rats born 3-5 weeks old were killed by neck dislocation, soaked in 75% alcohol for 3 minutes, and then transferred to an ultra-clean workbench for operation. Cut off the skin of the hind limbs, remove the leg muscles, and expose the femur. The external muscles of the femur are stripped clean, and the femur is cut off;

[0050] S2: Cut off the femoral head at both ends of the femur, use a 15ml syringe to absorb DMEM / F12 basal medium, blow out the femoral bone marrow, and rinse the bone marrow during the blowing process;

[0051] S3: Use a Pasteur pipette to blow the bone marrow 30 times to make a single-cell suspension, centrifuge through a centrifuge, collect the cells, and discard the supernatant;

[0052] S4: Add 9ml red blood cell lysate and lyse at 4 degrees Celsius for 3 minutes, then centrifuge through a centrifuge to collect the cells, discard the su...

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Abstract

The invention discloses a rat bone marrow derived mast cell culture method. The culture method comprises the following steps: killing SD young rats born for 3-5 weeks by a neck-removing method, soaking the SD young rats in 75% alcohol for disinfection for 3 minutes, then transferring the rats into an ultra-clean workbench for operation, cutting off hind limb skin, removing leg muscles, exposing femur, peeling off external muscles of the femur, and cutting off the femur; cutting off femoral heads at the two ends of the femur, sucking a DMEM / F12 basal culture medium by using an injector, blowingand beating out femoral marrow, and flushing the marrow cleanly in the blowing and beating process; blowing and beating the bone marrow for 30 times by using a Pasteur pipette to prepare a single-cell suspension, performing centrifuging by using a centrifuge, collecting cells, and discarding supernatant; and adding red blood cell lysate and carrying out lysis at 4 DEG C for 3 min. According to the method, the mast cell induction culture medium and the mast cell maturation promoting culture medium are used for culture and multiple times of centrifugal separation, so that the cultured mast cells are relatively high in purity, and the culture period is greatly shortened.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing rat bone marrow-derived mast cells. Background technique [0002] Mast cells are widely distributed around the microvessels under the skin and visceral mucosa, secrete a variety of cytokines, participate in immune regulation, express MHC molecules, B7 molecules, have APC function, express a large number of IgE Fc receptors, release allergic mediators, and have weak phagocytosis Function, like blood basophils, tissue cells with strong basophilic granules, past studies on mast cells have focused on their role in hypersensitivity diseases, but since the first discovery of mast cell defects in 2006 It is difficult for mice to induce skin transplantation tolerance, but infusion of bone marrow-derived MC to reconstitute MC can induce transplantation tolerance in normal mice, which proves the key role of mast cells in organ transplantation tolerance, but the ...

Claims

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Application Information

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IPC IPC(8): C12N5/0787
CPCC12N5/0642C12N2509/10C12N2509/00C12N2500/44C12N2501/22C12N2501/2303C12N2500/84
Inventor 范振峰孔令泽李慧贺永胜
Owner LABREAL BIOTECH KUNMING CO LTD
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