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Flow Cytometry (FC) Protocol

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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.


IF Diagram

FC diagram courtesy of Bitesize Bio. Click to enlarge.

Solution and reagents

  • 4% formaldehyde
  • 1%TritonX-100
  • 10% normal goat serum
  • Primary antibody
  • Secondary-fluorescence antibody

Sample preparation

Preparation for a sample of cell

  1. To suspend cells, centrifuge cells at 1000 RPM. The recommended concentration of cells is 1×106.
  2. Wash the cells by ice-cold PBS three times.

Preparation for tissue samples

  1. Shear and digest tissue mass by pancreatin or collagenase.
  2. Observe under microscope to single dispersed cell 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 and glutaraldehyde) and a denaturing reagent (e.g., 70% ethanol and 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. For intracellular proteins, fixation is needed.

  1. A 70% ethanol fixation can be performed for 12 hours at 4℃ (can be stored at -20℃ for a month) and 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 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.



**This is a suggested protocol and should be adjusted by the user accordingly**
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