First, the experimental preparation

1. Specimen preparation: 2. Minimize non-specific binding:

Second, apoptosis

1. Methods for detecting apoptosis: websites and others 2. PI staining

3. Annexin V method 4. TUNNEL method

Third, cytokines

1. Activated cytokines 2. CBA

Fourth, platelets

1. Activation 2. Activation detection

3. Reticulated platelets

Five, red blood cells

1. Reticulocyte 2. PNH

3. Fetal red blood cells

6. Oncology

1. DNA cell cycle 2. protein

3. Multidrug resistance 4. Minimal residual leukemia

Part 1 Specimen processing

1. Sample preparation during routine detection by flow cytometry

 (1) Direct immunofluorescence labeling

Take a certain amount of cells (about 1×10 6 cells/ml), add 50 μl of HAB to each tube, mix well, let stand for 1 minute or more at room temperature, and then directly immunize with fluorescein-conjugated antibody. Labeling reactions (such as double-labeled or multi-labeled staining, several antibodies labeled with different fluoresceins can be added at the same time). After incubation for 20-60 minutes, wash 1-2 times with PBS (pH 7.2-7.4), resuspend in buffer, and test on the machine. The method is simple and convenient to operate, and the result is accurate and easy to analyze, and is suitable for simultaneous determination of multiple parameters of the same cell group. Although the cost of the direct-labeled antibody reagent is high, it reduces the interference of strong non-specific fluorescence in the indirect labeling method, and thus is more suitable for the detection of clinical specimens.

 (two) indirect immunofluorescence labeling

Take a certain amount of cell suspension (about 1X106 cells/ml), first add a specific first antibody, wash away the unbound antibody after the reaction is complete, and then add the fluorescently labeled secondary antibody to form an antigen-antibody-antibody antibody complex. The FCM detects the fluorescence emitted by the fluorescein labeled on it. The method is low in cost, and the secondary antibody is widely used, and is mostly used for the detection of scientific specimens. However, since the secondary antibody is generally a polyclonal antibody, the specificity is poor, and the non-specific fluorescent background is strong, which easily affects the experimental results. Therefore, a negative or positive control should be added to the preparation of the specimen. In addition, because there are many steps in the inter-labeling method, the loss of cells is increased, and it is not suitable to measure specimens with a small number of cells.

Second, the method of minimizing non-specific binding

1. The concentration of the fluorescently labeled antibody should be appropriate, and if the concentration is too high, the background will increase due to an increase in non-specific interaction.

2. Prior to use of the primary antibody, the sample is incubated with excess protein, such as bovine serum albumin (BSA), non-fat dry cheese, or normal serum from the same host as the labeled secondary antibody. This step reduces the background by blocking the non-specific interaction of the first antibody with the cell surface or intracellular structure.

3. After the first antibody is used, the sample is incubated with 5% to 10% of normal serum from the same host and a secondary antibody as a label. This step reduces the interaction between the unwanted second antibody and the first antibody, cell surface or intracellular structure.

This step can be skipped by diluting the labeled antibody with serum from the same sample. This step is applicable in many ways, but sometimes it also results in the formation of an immunocomplex of the labeled secondary antibody and immunoglobulin in normal serum. Such complexes will preferentially bind to some cellular structures, or they will ultimately result in the loss of the desired antibody activity.

4. The use of the F(ab')2 fragment will allow the background to be determined by the binding of the first or second antibody to the full molecule of the FC receptor. Most of the F(ab')2 fragments of the second antibody are readily available. The F(ab')2 fragment of the first antibody is generally unusable or difficult to produce. Therefore, in the presence of NaN3, fresh tissue or cells should be preferentially added to the primary antibody when incubated with normal serum. In this case, even if all of the antibody molecules are used up in subsequent steps, the background influence determined by the FC receptor is no longer important.

5. Other: Cross-reactivity of labeled antibodies with other intrinsic immunoglobulins or other substances added to the experimental system may also have background effects. In order to reduce background, all labeled antibodies should be adsorbed during the multi-labeling process to avoid cross-reactivity of other species of proteins.

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