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Flow Cytometry Protocol

Flow Cytometry Protocol Page for Cell Analysis

Flow cytometry is a powerful technique used for the quantitative analysis of cell populations and the detection of specific cancer and cell biomarkers. This protocol outlines the key steps involved in performing flow cytometry, highlighting the use of antibodies for cell surface or intracellular marker detection.

Materials

  • Cell Suspension: Prepare a single-cell suspension of your sample.
  • Fluorescent Antibodies: Obtain specific fluorescently labeled antibodies targeting your markers of interest.
  • Buffer Solutions: Prepare and use appropriate buffers (e.g., PBS, FACS buffer).
  • Staining tubes for antibody incubation. 
  • FACS tubes or plates for sample analysis.
  • Flow Cytometer: Ensure the flow cytometer is calibrated and maintained.
  • Isotype Controls: Include appropriate isotype controls for antibody specificity.
  • Compensation Beads: Compensate for spectral overlap in the flow cytometer.

*Make sure to use personal protective equipment, such as gloves and lab coats.

Flow Cytometry of human Adipophilin on PBMC. Green: Isotype Control; Red: Adipophilin Monoclonal Antibody (ADFP/1365).

Flow Cytometric Analysis of Human HeLa cells using Smooth Muscle Actin Mouse Monoclonal Antibody (1A4) followed by Goat anti-Mouse IgG-CF488 (Blue); Isotype control (Red).

Flow cytometric analysis of PFA-fixed HeLa cells. AGO3Mouse Monoclonal Antibody (PCRP-AGO3-1C5) followed by goat anti-mouse IgG-CF488 (blue); isotype control (red).

Flow Cytometric Analysis of paraformaldehyde-fixed HepG2 cells using Albumin Monospecific Mouse Monoclonal Antibody (ALB/2356) followed by goat anti-Mouse IgG-CF488 (Blue); Isotype Control (Red).

Procedure

  1. Prepare a single-cell suspension of your sample by washing and resuspending in a suitable buffer.
  2. Aliquot your cell suspension into staining tubes. Add the appropriate fluorescently labeled antibodies to your samples. Follow the recommended antibody concentration and incubation time.
  3. Include isotype controls for each antibody used to assess background staining.
  4. Wash your stained cells to remove unbound antibodies. Centrifuge the cells and resuspend them in fresh buffer.
  5. Use compensation beads and single-stained controls to set up compensation.
  6. Load your stained and washed cell samples into FACS tubes or plates. Analyze your samples using the flow cytometer, following the instrument’s protocol for acquisition.

Analyze the acquired data using flow cytometry analysis software to identify and quantify target molecules. Flow cytometry provides data on cell populations and the expression levels of specific cell surface and/or intracellularly proteins.

Flow Cytometric Analysis of PFA-fixed HeLacells. ATF2 Mouse Monoclonal Antibody (PCRP-ATF2-1B4) followed bygoat anti-mouse IgG-CF488 (blue); unstained cells (red).

Flow Cytometric Analysis of PFA-fixed HeLa cells. Annexin A1 Mouse Monoclonal Antibody (ANXA1/1671) followed by goat anti-Mouse IgG-CF488 (Blue); Isotype Control (Red).

Flow Cytometric Analysis of PFA-fixed HeLa cells using Annexin A1 Mouse Monoclonal Antibody (ANXA1/3566) followed by goat anti-Mouse IgG-CF488 (Blue); Isotype Control (Red).

Flow Cytometric Analysis of HeLa cells using APEX Nuclease I Mouse Monoclonal Antibody (CPTC-APEX1-2). Goat anti-Mouse IgG-CF488 (Blue); Isotype Control (Red).

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