Flow Cytometry (FCM) is a single-cell quantitative analysis and sorting technique performed using a flow cytometer. Flow cytometry is a high-tech product resulting from the highly developed and comprehensive integration of monoclonal antibody and immunocytochemistry techniques, lasers, and computer science. It can effectively distinguish heterogeneous cell populations at the single-cell level. The detection objects include, but are not limited to, suspension cells, adherent cells, or single-cell suspensions isolated from solid tissues, as well as other biological particles.
01. Cell Surface Staining Procedure
1. Sample Preparation
1.1. Collect whole blood or tissue (spleen, lymph node, thymus, and bone marrow), and prepare a single-cell suspension using cell staining buffer (or PBS containing 1% BSA). For in vitro stimulated cells, directly suspend the stimulated cells in cell staining buffer (or PBS containing 1% BSA), and then proceed to Step 2.
1.2. Fill with cell staining buffer (or PBS containing 1% BSA), centrifuge the cell suspension at 300g for 5 minutes, and discard the supernatant.
2. Red Blood Cell Lysis
2.1. If red blood cell lysis is required (e.g., for spleen), dilute 10× red blood cell lysis buffer (ACK buffer) with deionized water to 1×, and place at room temperature. Resuspend the cells in 3 mL of 1× ACK buffer and incubate at room temperature for 2-3 minutes; if red blood cell lysis is not required, proceed directly to Step 3.
2.2. Add 10 mL of cell staining buffer (or PBS containing 1% BSA) to terminate red blood cell lysis, centrifuge the cell suspension at 300 × g for 5 minutes, and discard the supernatant.
2.3. Repeat the wash once, fill with cell staining buffer (or PBS containing 1% BSA) to 15 mL, centrifuge the cell suspension at 300 × g for 5 minutes, and discard the supernatant.
2.4. Count the cells, and prepare a cell suspension at 1 × 107/mL using cell staining buffer (or PBS containing 1% BSA). Add 100 μL of cell suspension to a 2 mL EP tube for later use.
3. Fc Receptor Blocking
Fc receptor blocking can reduce non-specific staining during the staining process.
For mouse samples, purified CD16/CD32 monoclonal antibody can bind to FcγRIII/II, blocking non-specific staining and reducing the background fluorescence of negative cells to the level of unlabeled cells. Add 0.5-1 μg of purified anti-mouse CD16/32 monoclonal antibody (E-AB-F0997A), and incubate at room temperature for 10 minutes.
For rat samples, an excess of purified Ig of the same source and isotype as the fluorescent antibody or serum of the same source can be used for blocking, or a commercial Fc receptor blocker can be used.
For human samples, EasyStain™ Human Fc Receptor Blocking Solution can be used as an FcR blocking agent. Add 5 μL of EasyStain™ Human Fc Receptor Blocking Solution. (E-CK-A171), and incubate at room temperature for 10 min.
4. Cell Staining
4.1. Add fluorescently labeled antibodies according to the recommended amount in the instructions, mix well, and incubate at 4°C in the dark for 30 minutes.
4.2. Add an 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 perform detection and analysis using a flow cytometer.
Precautions
1. Before using the antibody, centrifuge briefly to gather it at the bottom of the tube.
2. Fluorescently labeled antibodies should be stored at 4°C in the dark and should not be frozen.
3. During staining, fixation or delayed analysis may reduce the fluorescence signal of certain antibodies. To obtain better results, analysis should be performed immediately after staining.
02. Intracellular Cytokine Staining Procedure
1. Cell Preparation
1.1. Collect the cells. For cells that have been stimulated and blocked (refer to the literature for specific treatment protocols), add cell staining buffer (or PBS containing 1% BSA) to prepare a single-cell suspension, and adjust the cell concentration to approximately 1 × 107/mL.
1.2. Take 100 μL of cells/tube (approximately 1 × 106 cells), and add them respectively to the bottom of labeled EP tubes.
2. Fixation
2.1. If surface staining is required, first perform surface staining with appropriate antibodies according to the "Cell Surface Staining Procedure."
2.2. Resuspend the cells in the flow tube with 1× Fixation/Permeabilization buffer, and incubate at room temperature in the dark for 30 minutes.
2.3. Centrifuge at 300 × g for 5 minutes, and discard the supernatant.
3. Permeabilization
3.1. Resuspend the fixed cells with 1× Permeabilization Buffer, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
3.2. Repeat the wash once, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4. Intracellular Staining
4.1. Resuspend the cells in 100 μL of 1× Permeabilization Buffer, add the corresponding antibodies, mix well, and incubate at room temperature in the dark for at least 30 minutes.
4.2. Add 2 mL of 1× Permeabilization Buffer to resuspend the cells, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4.3. Add 2 mL of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, centrifuge at 300 × g for 5 minutes, and discard the supernatant.
4.4. Add 0.2 mL of cell staining buffer (or PBS containing 1% BSA) to resuspend the cells, and perform detection and analysis using a flow cytometer.
Precautions
1. When performing both cell surface and intracellular protein staining on the same cells, it is recommended to perform cell surface staining first, followed by fixation and permeabilization.
2. Isotype controls are recommended for intracellular staining.
