Flow Cytometry (FACS)

Flow Cytometry (FACS)

Flow cytometry is used for the separation, classification and quantitation of cells. Complex computerized instruments are used to pass a monocellular stream of cells, platelets or other microscopic particulate elements through a beam of laser light. The cells are categorized first by size and then computer analysed to sort the mixture of cellular elements into cell type by size. In addition, antibodies conjugated to fluorescent dyes are used to mark specific cell components. Each cell will display an appropriate fluorescent light emission, consistent with the total component presence in the cell. This emission is counted. Tabulation of counted data, in conjunction with size analysis, enables determination of relative percentages of each specific cellular subset for which antibody conjugates are utilised, even when the size of the cell is identical to other subset species. More information can be found here.

Reagents

  • PBS buffer: pH 7.4
  • Blocking Buffer: 0.5% BSA-PBS
  • Fix Buffer: 2-4% paraformaldehyde
  • Penetrating Buffer: 90% methanol

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.