Flow cytometry procedure

Flow cytometry (FCM) is a single-cell quantitative analysis and sorting technique using flow cytometry. Flow cytometry is a highly developed and comprehensively utilized high-tech product of monoclonal antibody and immunocytochemistry technology, laser and computer science, etc. It can effectively differentiate heterogeneous cell populations from the single-cell level, and the test objects include, but are not limited to, suspension cells, adherent cells, or single-cell suspensions and other biological particles separated from solid tissues.

Cell surface staining steps
1. Sample preparation
1.1 Collect whole blood or tissue (spleen, lymph nodes, thymus, and bone marrow) and make a single-cell suspension with cell staining buffer (or PBS with 1% BSA). For in vitro stimulated cells, suspend the stimulated cells directly in cell staining buffer (or PBS containing 1% BSA) and proceed to step 2.
1.2 Fill up the cell staining buffer (or PBS containing 1% BSA), centrifuge the cell suspension at 300g for 5 minutes and discard the supernatant.

2. Erythrocyte lysis
2.1 If erythrocytes need to be lysed (e.g., spleen), dilute 10x erythrocyte lysate (ACK buffer) to 1x with deionized water and allow to come to room temperature. Resuspend cells in 3mL of 1x ACK buffer and incubate at room temperature for 2-3 minutes; if no lysis of erythrocytes is required, proceed directly to step 3,.
2.2 Terminate erythrocyte lysis by adding 10mL of cell staining buffer (or PBS with 1% BSA), centrifuge cell suspension at 300g for 5 minutes and discard supernatant.
2.3 Repeat the wash, fill up the cell staining buffer (or PBS containing 1% BSA) to 15mL, centrifuge the cell suspension at 300g for 5 minutes, and discard the supernatant.
2.4 For cell counting, make a 1x107/mL suspension of cells with cell staining buffer (or PBS containing 1% BSA). Add 100μL of cell suspension to a 2mL EP tube and set aside.

3. Sealing of Fc receptors
Blocking the Fc receptor reduces non-specific staining during the staining process.
For mouse samples, purified CD16/CD32 Monoclonal Antibody binds to FcγRIII/II, sealing off non-specific staining and reducing the background fluorescence of negative cells to the level of unlabeled cells. Add 0.5~1μg of pure anti-mouse CD16/32 monoclonal antibody and incubate for 10 minutes at room temperature.
For rat samples, blocking can be performed with an excess of purified Ig of the same source and isoform as the fluorescent antibody or serum of the same source, or with a commercial Fc receptor blocker.
For human samples, purified CD16 monoclonal antibody can be used as a blocking FCR blocker. Add 1 μg of pure anti-human CD16 monoclonal antibody and incubate for 10 min at room temperature.

4. Cell staining
4.1 Add fluorescently labeled antibody according to the recommended dosage in the instruction manual, mix well and incubate at 4°C, protected from light for 30 minutes.
4.2 Add appropriate amount of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, centrifuge the cell suspension at 300 g for 5 minutes, and discard the supernatant.
4.3 Add 0.2 mL of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, and detect and analyze by flow cytometry.
Precautions
1. Centrifuge the antibody quickly to bring it to the bottom of the tube before use.
2. Fluorescently labeled antibodies should be stored at 4°C away from light, not frozen.
3. When staining, fixation or delayed analysis may reduce the fluorescence signal of some antibodies. For better results, the antibody should be analyzed immediately after staining.

Steps for staining intracellular cytokines
1. Cell preparation
1.1 Collect cells. Cells after stimulation and blocking treatment (please refer to the literature for specific treatment protocols to be carried out), add cell staining buffer (or PBS containing 1% BSA) to make a single-cell suspension, and adjust the concentration of cells to about 1 × 107/mL.
1.2 Take 100 μL cells/tube (about 1 × 106 cells) and add them to the bottom of well-labeled EP tubes respectively.
2. Fixation
2.1 If surface staining is required, select the appropriate antibody for surface staining according to the “Cell Surface Staining Procedure”.
2.2 Resuspend the cells in the flow-through tube with 1× cell fixation/permeabilization buffer and incubate for 30 minutes at room temperature away from light.
2.3 Centrifuge at 300 g for 5 minutes and discard the supernatant.
3. Membrane Breaking
3.1 Suspend the fixed cells with 1× Cell Membrane Breaking Buffer (Permeabilization Buffer), centrifuge at 300g for 5 minutes, and discard the supernatant.
3.2 Repeat the wash, centrifuge at 300g for 5 minutes, and discard the supernatant.
3.3 Add 1 mL of 1×Cell Membrane Breaking Buffer (Permeabilization Buffer) and incubate for 30 minutes at 4℃ away from light; centrifuge at 300g for 5 minutes and discard the supernatant.

4. Intracellular staining
4.1 Resuspend the cells with 100 μL of 1×Cell Membrane Breaking Buffer (Permeabilization Buffer), add the corresponding antibody, mix well, and incubate at least 30 minutes at 4℃ away from light.
4.2 Add 2 mL of 1×Cell Membrane Breaking Buffer (Permeabilization Buffer) to resuspend the cells, centrifuge at 300g for 5 minutes, and discard the supernatant.
4.3 Add 2 mL of Cell Staining Buffer (or PBS containing 1% BSA) to resuspend cells, centrifuge at 300g for 5 minutes, and discard supernatant.
4.4 Add 0.2 mL of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, and detect and analyze by flow cytometry.
Precautions
1. For both cell surface and intracellular proteins in the same cell, it is recommended to do cell surface staining first, then fixation and membrane breaking.
2. Intracellular staining is recommended to use isotype control.

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