Fluorescence activated cell sorting

Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry used to sort and analyze a heterogeneous mixture of cells into distinct subgroups based on each cell's specific light-scattering and fluorescence characteristics (from specific markers). group. The technique can be used to isolate cell populations for a huge number of measurable parameters - from simple surface immunophenotypes to metabolic function, cell cycle status, redox status, and DNA content analysis, to name a few. FACS has been widely used in biomedical research as well as clinical diagnosis and treatment^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

Cells for flow cytometry analysis typically come from 3 main sources: Adherent or suspension culture; Samples cryopreserved; Whole blood or buffy coat solid tissues such as bone marrow, spleen, intestine, etc.

Regardless of the source, the final cell preparation should be: 1. Uniform single-cell suspension free of clumps and dead cell debris; 2. Density is 10⁶-10⁷ cells per mL; 3. Suspend in appropriate staining buffer.

PBMCs isolated from whole blood by Ficoll gradient centrifugation or RBC lysis of whole blood or non-adherent cultured cells can be used for flow cytometric analysis. Adherent cultured cells or cells present in solid organs should first be prepared as single cell suspensions prior to flow analysis using enzymatic digestion or mechanical dissociation of the tissue, respectively. Mechanical filtration should be followed to avoid unnecessary clogging of the instrument or lower quality flow data.

• Direct immunostaining of surface antigen

1. Prepare a single cell suspension using an appropriate protocol and adjust the cell concentration to 10⁷ cells/mL in staining buffer.

2. Distribute 100 µL of cell suspension into as many stained tubes as desired (unstained controls, compensated controls, optional isotype and FMO controls, and test samples).

3. Add the optimized dilution of antibody to the corresponding tube and incubate at 4 °C (on ice) for 30 min in the dark.

4. Wash cells once with ice-cold PBS at 300-400 × g, then resuspend in 100-200 μL FACS buffer/PFA fixation buffer.

5. Store in the dark at 4°C, preferably within 24 hours of acquisition.

• Indirect immunostaining of surface antigen

1. Prepare a single cell suspension using an appropriate protocol and adjust the cell concentration to 10⁷ cells/mL in staining buffer.

2. Distribute 100 µL of the cell suspension into as many stained tubes as desired (unstained controls, compensated controls, optional isotype and FMO controls, and test samples).

3. Add the optimized dilution of the primary antibody to the corresponding tube and incubate at 4°C (on ice) for 30 min.

4. Wash cells once with ice-cold PBS at 300-400 × g and resuspend in 100 μL staining buffer.

5. Add the specific secondary antibody at an appropriate dilution and incubate the cells at 4°C (on ice) for 30 min in the dark.

6. Wash the cells once with 300-400×g of cold PBS, then resuspend in 100-200 μL FACS buffer/PFA fixation buffer.

7. Store in the dark at 4°C, preferably within 24 hours of acquisition.

• General immunostaining procedure for intracellular antigens-methanol permeabilization

1. Surface staining.

2. Separate the stained cells at a concentration of 10⁷ cells/mL in 0.5-4% PFA. Prepare unstained aliquots for intracellular staining controls.

3. Fix the cells on ice for 10-30 min, away from light.

4. Wash off the fixative at 300-400 × g, then slowly add ice-cold 100% methanol and vortex gently.

5. Incubate the cells on ice for 30 minutes, away from light.

6. Wash methanol at 400-500 × g and resuspend cells in 100 µL staining buffer.

7. Distribute 100 µL of the cell suspension into as many stained tubes as desired (unstained controls, compensated controls, optional isotype and FMO controls, and test samples).

8. Add the optimized dilution of the primary antibody to the corresponding tube and incubate at 4°C (on ice) for 30 min.

9. Wash cells once with ice-cold PBS at 400-500 × g and resuspend in 100 μL staining buffer.

10. Add the specific secondary antibody at an appropriate dilution and incubate the cells at 4°C (on ice) for 30 min in the dark.

11. Wash cells once with 400-500 × g of cold PBS and resuspended in 100-200 μL FACS buffer/PFA fixation buffer.

12. Store in the dark at 4°C, preferably within 24 hours of acquisition.

• Dye efflux staining-propidium iodide (PI) staining

1. Harvest cells and wash once with PBS.

2. Resuspend cells in staining buffer at a concentration of 10⁷ cells/mL.

3. Add 100 μl PI stain to every 5 μl cell suspension, mix gently and let it stay in the dark for 1 minute.

4. Wash off the dye and resuspend the cells in an appropriate volume of staining buffer.

• DNA content or cell cycle analysis

1. Harvest cells and wash once in PBS.

2. Resuspend cells in staining buffer at a concentration of 10⁷ cells/mL.

3. Aliquot 500 μL cells into separate tubes (pre-chilled) and gently vortex to add ice-cold 70% ethanol dropwise.

4. Store the cells on ice for 1 hour.

5. Wash cells twice at 400-500 × g in PBS for 10 min.

1.Specialized gradient media have been formulated to enrich for different granulocyte populations.

2. If the cells are stained in a 96 well U- or V-bottom plate, washing procedure should be set up first for maximum removal of unbound primary antibodies.

3. In the case of staining for secreted cytokines, add brefeldin A or monensin during the incubation period at the concentration recommended by the manufacturer.

4. Set aside some unstimulated aliquots for the unstained control and stained baseline controls.