Surface Marker Analysis by Flow Cytometry

Introduction

Flow cytometry is a technique for counting particles using electronic detection apparatus, and is often used to collect quantitative information about cell populations. The technique involves labeling cells with a fluorescent marker, and suspending cells in a stream of fluid which passes through, and is measured by a fluorescence measuring station.

Required Materials

Cells

  • Cells in suspension

Reagents and Equipment

  • 0.1% BSA in PBS (Staining Medium)
  • Fluorescently labeled antibody
  • Flow cytometer
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Titrating Antibodies

Determining the Optimal Concentration of Antibody for Flow Cytometry

  1. Dilute labeled antibodies for the appropriate antigens to be detected in Staining Medium. Make dilutions of all antibodies at x1, x2, x5, x10, x20, x40, x80 and x100.
  2. Prepare the cells that express the antigen to be analyzed.
  3. Count the number of cells.
  4. Use 1 × 106 cells for each dilution. Smaller numbers of cells ranging from 50,000 to 100,000 may work as well.
  5. Centrifuge cells at 300 × g for 5 minutes at 4°C and discard the supernatant.
  6. Add 5 μL of antibody from each dilution into separate sample tubes containing cells.
  7. Prepare negative controls of cells that have not been stained with antibody, and cells stained with an isotype control.
  8. Mix well and incubate cells on ice for 25-30 minutes.
  9. If primary antibodies are not directly conjugated to fluorescent tags, carry out the second step incubation with secondary antibody tagged to a fluorescent tag.
  10. Wash with 10 mL of Staining Medium. Discard the supernatant and resuspend the cells in 0.5 mL of Staining Medium.
  11. Analyze the cells by flow cytometry.

Note: Use the same cell number in every experiment. Starting with larger numbers of cells is preferred since setting up parameters during flow cytometry analysis takes time and collecting >10,000 events produces more reliable data.
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One-Step Staining with Fluorescently Labeled Antibodies

One-Step Staining with Fluorescently-labeled Antibody

  1. Trypsinize cells and add Staining Medium. Transfer the cells to a conical tube and centrifuge at 300 × g, 4°C for 5 minutes. Discard the supernatant.
  2. Add 5 μL of diluted primary antibody conjugated to a fluorescent tag to the cell pellet.
  3. Flick the tube to resuspend the cell pellet. Mix well and incubate on ice for 25-30 minutes.
  4. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C for 5 minutes.
  5. Discard the supernatant and resuspend the cells with 0.5 mL of Staining Medium.
  6. Filter the cell suspension through FACS filter tubes before analysis or sorting the cells by flow cytometry.

Note: For negative controls, prepare cells that have not been stained with antibody, and cells stained with an isotype control.
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Two-Step Staining with Biotinylated Antibodies

Two-step Staining with Biotinylated Antibody

  1. Trypsinize cells and add Staining Medium. Transfer the cells to a conical tube and centrifuge at 300 × g, 4°C for 5 minutes. Discard the supernatant.
  2. Add 5 μL of appropriately diluted biotinylated primary antibody.
  3. Flick the tube to resuspend the cell pellet. Mix well and incubate on ice for 25-30 minutes.
  4. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C for 5 minutes.
  5. Discard the supernatant. Add diluted streptavidin secondary antibody conjugated to a fluorescent tag.
  6. Mix well and incubate the cells on ice for 25-30 minutes.
  7. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C for 5 minutes.
  8. Discard the supernatant and resuspend cells with 0.5 mL of Staining Medium.
  9. Filter the cell suspension through FACS filter tubes before analysis or sorting the cells by flow cytometry.


Note: For negative controls, prepare cells that have not been stained with antibody, and cells stained with an isotype control.

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LT162                   17-Mar-2011