Flow Cytometry (FC) Protocol

Instructions

Flow Cytometry is a laser-based technique used to rapidly analyze the characteristics of cells and other particles. FC can analyze tens of thousands of cells while simultaneously measuring multiple parameters from a single cell. Fluorescently labeled cells flow past a laser, emitting a measurable fluorescent signal.

FC diagram courtesy of Bitesize Bio.

Solution and reagents

• 4% formaldehyde
• 0.2% TritonX-100
• 10% normal goat serum
• Primary antibody
• Secondary-fluorescence antibody

Sample preparation

Preparation for cells

1. For suspension, collect by centrifugation at 1000 RPM. For adherant cells, detatch by trypsinization, then centrifuge. The recommended number of cells is 1×10⁶.
2. Wash the cells by ice-cold PBS three times.

Preparation for tissues

1. Shear and digest tissue mass by pancreatin or collagenase.
2. Observe under a microscope until a single-cell dispersion is obtained and terminate digestion.

Pancreatin is often used to digest mesenchymal tissue, while collagenase works well for collagen structures. Generally, Pancreatin digestion can be performed for 20-60 min. For collagenase treatment, 4-48 hours is needed.

Fixation and permeabilization

Fixation and permeabilization of samples are critical steps that can affect experimental success. A cross-linking reagent (e.g., 4% formaldehyde, 10% formalin, glutaraldehyde) or a denaturing reagent (e.g. 70% or 90% ethanol) are often used as a fixative. Denaturing reagents break cell membranes, while cross-linking agents work to fixate cellular proteins. If fixation is performed with a denaturing reagent, permeabilization is not needed to label intracellular proteins. If fixation is performed by cross-linking, permeabilization by 0.2% TritonX-100 is required to label intracellular proteins. Permeabilization is not required if the target protein is a membrane-bound protein.

1. A 70% ethanol fixation can be performed for 12 hours at 4℃ (can be stored at -20℃ for a month) or a 4% formaldehyde fixation can be performed for 15 min at RT. For permeabilization, add 0.2% TritonX-100 for 5min (can be stored at 4℃ for one week).
2. Centrifuge at 1000 RPM and wash cells by PBS three times.

Blocking

Blocking reduces non-specific binding between antibodies and other proteins that are not specific to the antibody. The recommended blocking solution is normal goat serum (note: the species must be consistent with the secondary antibody).

Antibody incubation

1. Prepare primary antibody solution by referring to the manufacturer recommended dilution ratio.
2. Prepare a negative control at the same time by adding a normal rabbit or mouse IgG antibody. Incubate samples with primary antibody at 4℃ overnight. 
3. Wash cells with PBS three times.
4. Prepare secondary antibody solution by referring to the manufacturer recommend dilution ratio. Do not expose to light.
5. Incubate secondary antibody at 4℃ for 30 min.
6. Wash cells with PBS three times.
7. Re-suspend cells with 500μl of PBS .

Detection

Keep the sample away from light and detect fluorescence signal as soon as possible with a flow cytometer.