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Flow Cytometry (FACS)

Flow Cytometry is a method used for the separation, classification and quantification of cells. Samples are suspended in fluid, and are then focused via computerised mechanisms into a mono-cellular stream which is passed through a beam of laser light. Through the measurement of scattered light, cells can be categorised by size, and subsequently by cell type. Antibodies conjugated to both fluorescent dyes and fluorescent intercalating agents are often used to mark specific cell components; targets will display a fluorescent light emmision which is proportional to the total target presence in the cell. The staining procedure involves making single-cell suspensions from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with or without fluorescently-labelled antibodies and analysed using a flow cytometer. Size analysis, in conjunction with counted fluorescent emission, enables the determination and characterisation of specific cellular subsets, even when the size of the cell is identical to other subset species. This provides a simultaneous multi-parameter analysis of single cells for the characterisation and definition of different cell types in heterogeneous cell populations. More information can be found here.

See below for a general protocol, and recommendations.

Reagents

  • PBS Buffer: pH 7.4
  • Blocking Buffer: 0.5% BSA-PBS
  • Fix Buffer: 2-4% paraformaldehyde
  • Penetrating Buffer: 90% methanol
  • Primary Antibody
  • Secondary Antibody (this is not required if the sample was directly labelled with a conjugated primary antibody)

Procedure

  1. Cell Collection: Collect cells and adjust the cell concentration to 1-5 x 106 cells/ml.
  2. Wash and Centrifuge: Add 2 ml blocking buffer, then shake slightly and centrifuge at 1500-2000 rpm for 5 min.
  3. Cell Fixation: Discard the supernatant, then fix cells in 1 ml fix buffer and incubate at room temperature for 10 min.
  4. Wash and Centrifuge: Centrifuge at 1500-2000 rpm for 5 min, then wash cells once with 1 ml blocking buffer and centrifuge again at the same condition.
  5. Permeabilisation: Discard the supernatant, add 1 ml precooled penetrating buffer and incubate at room temperature for 10 min (If the target is extracellular, skip this step).
  6. Wash and Centrifuge: Centrifuge at 1500-2000 rpm for 5 min, then wash cells once with 2 ml blocking buffer and centrifuge again at the same condition.
  7. Blocking: Incubate cells in blocking buffer for 30 min at room temperature.
  8. Incubate Primary Antibody: Add primary antibody at 0.025 mg/ml and incubate for 90 min at room temperature.
  9. Wash and Centrifuge: Centrifuge at 1500-2000 rpm for 5 min, then wash cells once with 2 ml blocking buffer and centrifuge again in the same conditions. Repeat this once.
  10. Incubate Secondary Antibody: Incubate with conjugated secondary antibody for 40 min at room temperature (For direct labelling using a conjugated primary antibody, skip this step).
  11. Wash and Centrifuge: Centrifuge at 1500-2000 rpm for 5 min, then wash cells once with 2 ml blocking buffer and centrifuge again at the same condition.
  12. FC analysis: Re-suspend cells in 1 x PBS and analyse on flow cytometer.