CN110672500B - Th detection method of non-heparin anticoagulant fluid sample - Google Patents
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Abstract
The invention discloses a Th detection method of a non-heparin anticoagulant fluid sample, which comprises the steps of processing the non-heparin anticoagulant sample by using lymphocyte separation fluid to obtain Peripheral Blood Mononuclear Cells (PBMC), removing the influence of the anticoagulant on cell activation and cytokine secretion, and simultaneously adding serum to provide enough nutrient substances for cells in a stimulation/blocking stage; after antibody staining and fixed membrane rupture, the kit can be successfully used for detecting Th cell factors, thereby expanding the range of Th detection sample types. The method can enable researchers or detection personnel to fully utilize the sample to carry out Th detection, avoids sample waste and repeated collection caused by collection of the non-heparin anticoagulant sample, and greatly improves efficiency.
Description
Technical Field
The invention relates to a Th detection method of a non-heparin anticoagulant fluid sample.
Background
The expression Th1, Th2 and Th17 refers to T helper cells (strictly Th0 cells) that have the ability to differentiate into Th1, Th2 and Th17 under various physiological and pathological conditions. In a resting state (i.e., without any stimulation, such as a normal physiological state in humans), Th0 has a very weak ability to differentiate into Th1, Th2 and Th17, so peripheral blood contains only very small amounts of Th1, Th2 and Th17 cells, and IFN-gamma, IL-4 and IL-17A are detected to a very small extent. When the Th cell is stimulated by external factors (such as stimulin, pathogen, etc.), Th0 will differentiate towards Th1, Th2 or Th17, and the differentiation depends on the kind of cytokine. In this case, too, IFN-. gamma.IL-4 or IL-17A was detected in a large amount. The experimental tests of Th1, Th2 and Th17 actually tested the ability of Th cells to respond to stimulation with stimulants.
The stimulants usually selected are Phorbol 12-Myrristate 13-Acetate (PMA, Phorbol ester) and Ionomycin (Ionomycin). PMA is an activator of PKC (protein kinase C), and PKC can activate phosphorylation of a plurality of downstream protein kinases to form a cascade reaction, so that expression of a plurality of proteins is caused, and activation of T cells is further caused. In cells, PKC can be activated by the interaction of DAG (diacylglycerol) and Ca2+, so that in T cells, PKC can be further activated in the presence of Ionomycin (a transporter of Ca2+, which transports Ca2+ in organelles into cytoplasm). It can be seen that PMA activates T cells in concert with Ionomycin.
Activated T cells can secrete a variety of cytokines out of the cell, whereas flow cytometry can only detect intracellular antigens, so cytokines should be blocked intracellularly. Cytokines are synthesized in the golgi apparatus and certain proteins are secreted extracellularly in soluble form by endoplasmic reticulum trafficking. The transfer pathway of the cell factor can be cut off by destroying the Golgi apparatus, and the secretion of the cell factor is blocked. The blocking agents usually used are Brefeldin A (BFA, Brefeldin A) and/or Monensin (Monensin).
The traditional Th detection method is only limited by heparin anticoagulated whole blood, while other types of anticoagulated samples (such as EDTA anticoagulated blood and sodium citrate anticoagulated blood) and the like influence the activation of Th cells and the secretion of cytokines, and the cytokines cannot be detected, so that the measurable sample types are limited. For researchers and detection personnel, the problem of sample waste or the need of re-collection is caused, and the efficiency is greatly influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Th detection method of a non-heparin anticoagulant fluid sample, aiming at solving the problems that the activation of Th cells and the secretion of cytokines can be influenced when the non-heparin anticoagulant fluid sample is detected in Th, the cytokines cannot be detected, the measurable sample type is limited, and the sample is wasted or needs to be collected again.
In order to achieve the purpose, the invention provides the following technical scheme: a Th detection method of a non-heparin anticoagulant fluid sample comprises the following steps:
the method comprises the following steps: separating Peripheral Blood Mononuclear Cells (PBMCs) with lymphocyte separating medium, and resuspending the precipitate in culture medium containing 10% fetal calf serum to obtain cell concentration of 1 × 107Taking 250 mu l of PBMCs into a flow tube, adding 1 mu l of PMA/Ionomycin Mixture (250 x) and 1 mu l of BFA/Monensin Mixture (250 x), taking a sample only containing the PBMCs as a control, mixing uniformly, incubating at 37 ℃ for 4-6 hours, taking out every 1-2 hours, shaking and mixing uniformly;
step two: taking 100 mul of cell suspension from the sample tube and the control tube to a new flow tube, adding corresponding flow antibodies, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step three: adding 100 μ l FIX & PERM mediumA into each tube, shaking, mixing, and incubating for 15 min at room temperature in dark place;
step four: diluting 10 XFlow Cytometry settling Buffer to 1X with distilled water, adding 2ml of precooled 1 XFlow Cytometry settling Buffer per tube, centrifuging at 300 Xg for 5 minutes, and discarding the supernatant;
step five: adding 100 mul FIX & PERM Medium B and corresponding flow type antibody into each tube, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step six: 2ml of 1 Xflow Cytometry stabilizing Buffer was added to each tube, and centrifuged at 300 Xg for 5 minutes, and the supernatant was discarded.
Step seven: adding 500 μ l of 1 Xflow Cytometry stabilizing Buffer into each tube for resuspension, and detecting on a machine; or adding 500. mu.l of 1-4% paraformaldehyde for resuspension, and detecting at 2-8 deg.C in 24 hr under dark condition.
Further, for Human blood samples, Human lymphocyte separation fluid was used in step one, Anti-Human CD3, FITC and 5. mu.l Anti-Human CD8 α, PerCP-Cy5.5 in step two, and Anti-Human IFN- γ, PE and Anti-Human IL-4, APC in step five.
Further, for the Mouse blood sample, the Mouse lymphocyte separation solution is adopted in the first step, the flow antibody in the second step is 5 μ l of Anti-Mouse CD3 epsilon, FITC and 5 μ l of Anti-Mouse CD4, PerCP-Cy5.5, and the flow antibody in the fifth step is 5 μ l of Anti-Mouse IFN-gamma, PE.
Further, for Rat blood samples, Rat lymphocyte separation solution is used in the first step, flow type antibodies in the second step are 5 μ l of Anti-Rat CD3, FITC and 5 μ l of Anti-Rat CD8 alpha, PerCP-Cy5.5, and flow type antibodies in the fifth step are 5 μ l of Anti-Rat IFN-gamma, efluor 660.
The invention has the beneficial effects that: treating a non-heparin anticoagulated sample with a lymphocyte separation medium to obtain Peripheral Blood Mononuclear Cells (PBMC), removing the effect of the anticoagulant on cell activation and cytokine secretion, and adding serum to provide sufficient nutrients to the cells during the stimulation/blocking phase; after antibody staining and fixed membrane rupture, the kit can be successfully used for detecting Th cell factors, thereby expanding the range of Th detection sample types.
The method can enable researchers or detection personnel to fully utilize the sample to carry out Th detection, avoids sample waste and repeated collection caused by collection of the non-heparin anticoagulant sample, and greatly improves efficiency.
Drawings
Fig. 1 is a diagram of the Th detection result of embodiment 1 of the present invention:
FIG. 1.1 shows the Th assay results of human EDTA anticoagulated whole blood:
A0: unpimulated human EDTA anticoagulant derived PBMCs stain IFN- γ;
A1: PMA/Ionomycin stimulated and BFA/Monensin blocked human EDTA anticoagulant-derived PBMCs stained IFN- γ.
FIG. 1.2 shows the Th assay results of mouse EDTA anticoagulated whole blood:
A0: unstimulated mouse EDTA anticoagulant derived PBMCs stain IFN-gamma;
A1: PMA/Ionomycin stimulated and BFA/Monensin blocked mouse EDTA anticoagulant derived PBMCs stained IFN- γ.
FIG. 1.3 shows the Th test results of rat EDTA anticoagulated whole blood:
a0: unstimulated rat EDTA anticoagulant derived PBMCs stain IFN- γ;
a1: PMA/Ionomycin stimulated and BFA/Monensin blocked rat EDTA anticoagulant-derived PBMCs stained for IFN- γ.
Detailed Description
Example 1
A Th detection method of a non-heparin anticoagulant fluid sample comprises the following steps:
the method comprises the following steps: separating Peripheral Blood Mononuclear Cells (PBMCs) with lymphocyte separating medium, and resuspending the precipitate in culture medium containing 10% fetal calf serum to obtain cell concentration of 1 × 107Taking 250 mu l of PBMCs into a flow tube, adding 1 mu l of PMA/Ionomycin Mixture (250 x) and 1 mu l of BFA/Monensin Mixture (250 x), taking a sample only containing the PBMCs as a control, mixing uniformly, incubating at 37 ℃ for 4-6 hours, taking out every 1-2 hours, shaking and mixing uniformly;
step two: taking 100 mul of cell suspension from the sample tube and the control tube to a new flow tube, adding corresponding flow antibodies, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step three: adding 100 μ l FIX & PERM mediumA into each tube, shaking, mixing, and incubating for 15 min at room temperature in dark place;
step four: diluting 10 XFlow Cytometry settling Buffer to 1X with distilled water, adding 2ml of precooled 1 XFlow Cytometry settling Buffer per tube, centrifuging at 300 Xg for 5 minutes, and discarding the supernatant;
step five: adding 100 mul FIX & PERM Medium B and corresponding flow type antibody into each tube, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step six: 2ml of 1 Xflow Cytometry stabilizing Buffer was added to each tube, and centrifuged at 300 Xg for 5 minutes, and the supernatant was discarded.
Step seven: adding 500 μ l of 1 Xflow Cytometry stabilizing Buffer into each tube for resuspension, and detecting on a machine; or adding 500. mu.l of 1-4% paraformaldehyde for resuspension, and detecting at 2-8 deg.C in 24 hr under dark condition.
For Human blood samples, Human lymphocyte separation was used in step one, flow antibodies were 5 μ l anti-Human CD3, FITC and 5 μ l anti-Human CD8 α, PerCP-Cy5.5 in step two, and 5 μ l anti-Human IFN-. gamma., PE and 5 μ l anti-Human IL-4, APC in step five. As can be seen from FIG. 1.1, after the human EDTA anticoagulated whole blood sample is treated by the above method, compared with the unstimulated control, after PMA/Ionomycin stimulation and BFA/Monensin blocking, significant IFN-gamma cytokine secretion can be detected.
For the Mouse blood samples, the Mouse lymphocyte separation solution is adopted in the first step, the flow-type antibodies in the second step are 5 mu l of Anti-Mouse CD3 epsilon, FITC and 5 mu l of Anti-Mouse CD4, PerCP-Cy5.5, and the flow-type antibodies in the fifth step are 5 mu l of Anti-Mouse IFN-gamma and PE. As can be seen from FIG. 1.2, after the mouse EDTA anticoagulated whole blood sample is treated by the above method, compared with the unstimulated control, after PMA/Ionomycin stimulation and BFA/Monensin blocking, significant IFN-gamma cytokine secretion can be detected.
For Rat blood samples, the Rat lymphocyte separation solution was used in the first step, the flow antibody in the second step was 5. mu.l of anti-Rat CD3, FITC and 5. mu.l of anti-Rat CD8 α, PerCP-Cy5.5, and the flow antibody in the fifth step was 5. mu.l of anti-Rat IFN-. gamma., efluor 660. As can be seen from FIG. 1.3, significant IFN-. gamma.cytokine secretion was detected after PMA/Ionomycin stimulation and BFA/Monensin blocking after treatment of rat EDTA-anticoagulated whole blood samples as described above, compared to unstimulated controls.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (4)
1. A Th detection method of a non-heparin anticoagulant fluid sample is characterized by comprising the following steps:
the method comprises the following steps: separating Peripheral Blood Mononuclear Cells (PBMCs) with lymphocyte separating medium, and resuspending the precipitate in culture medium containing 10% fetal calf serum to obtain cell concentration of 1 × 107Taking 250 mu l of PBMCs into a flow tube, adding 1 mu l of PMA/Ionomycin Mixture (250 x) and 1 mu l of BFA/Monensin Mixture (250 x), taking a sample only containing the PBMCs as a control, mixing uniformly, incubating at 37 ℃ for 4-6 hours, taking out every 1-2 hours, shaking and mixing uniformly;
step two: taking 100 mul of cell suspension from the sample tube and the control tube to a new flow tube, adding corresponding flow antibodies, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step three: adding 100 μ l FIX & PERM Medium A into each tube, shaking, mixing, and incubating at room temperature in dark for 15 min;
step four: diluting 10 XFlow Cytometry settling Buffer to 1X with distilled water, adding 2ml of precooled 1 XFlow Cytometry settling Buffer per tube, centrifuging at 300 Xg for 5 minutes, and discarding the supernatant;
step five: adding 100 mul FIX & PERM Medium B and corresponding flow type antibody into each tube, shaking and uniformly mixing, and incubating for 15 minutes at room temperature in a dark place;
step six: adding 2ml of 1 Xflow Cytometry stabilizing Buffer into each tube, centrifuging for 5 minutes at 300 Xg, and discarding the supernatant;
step seven: adding 500 μ l of 1 Xflow Cytometry stabilizing Buffer into each tube for resuspension, and detecting on a machine; or adding 500. mu.l of 1-4% paraformaldehyde for resuspension, and detecting at 2-8 deg.C in 24 hr under dark condition.
2. The method of claim 1, wherein for Human blood samples, the flow-through antibodies in step one are 5 μ l Anti-Human CD3, FITC and 5 μ l Anti-Human CD8 α, PerCP-cy5.5, and the flow-through antibodies in step five are 5 μ l Anti-Human IFN- γ, PE and 5 μ l Anti-Human IL-4, APC.
3. The method of claim 1, wherein the Mouse lymphocyte separation solution is used in the first step, the flow-type antibody in the second step is 5 μ l of Anti-Mouse CD3 epsilon, FITC and 5 μ l of Anti-Mouse CD4, PerCP-Cy5.5, and the flow-type antibody in the fifth step is 5 μ l of Anti-Mouse IFN-gamma, PE.
4. The method of claim 1, wherein the Rat blood sample is treated with Rat lymphocyte separation solution in the first step, and the flow-type antibody in the second step is 5 μ l of Anti-Rat CD3, FITC, 5 μ l of Anti-Rat CD8 α, PerCP-Cy5.5, and the flow-type antibody in the fifth step is 5 μ l of Anti-Rat IFN- γ, efluor 660.
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