Titration of Antibodies – Flow Cytometry Staining Protocol

Methods and Principles from our Scientific Staff

Background

Titration is the process of identifying the correct concentration of antibody to use for a given assay. This ensures the antibody performs within acceptable parameters including reducing negative effects and waste. Titration for flow cytometry is used to determine the antibody amount and concentration resulting in the highest signal of the positive population along with the lowest signal of the negative population. It is also the best way to eliminate nonspecific binding of an antibody.

Why Do It?

The precise amount of an antibody is essential to proper titration.

Too much antibody will result in an increase in the background non-specific (or low affinity) binding, reducing the sensitivity of the fluorescent measurement.

Too little antibody will result in a decrease in the positive signal, again reducing the sensitivity of the fluorescent measurement.

Figure 1. (Left) As concentration of antibody increases there is greater separation of positive and negative cells based on fluorescence intensity.

Leinco Product C119, Anti-Human CD8 (Clone: UCHT-4) – FITC

How To Do It

Titration experiments are easy to perform, and there are several papers that discuss this process1,2. This protocol has been optimized for 1×106 cells in 50 µl final volume. All staining is performed on ice, in the dark, for 20 minutes. Identify the 1x concentration, which is the manufacturer’s recommended concentration for using their antibody in a given assay. These concentrations can either be a volume (10 µl per 100 µl) or a concentration (0.1µg/µl). Based on the starting concentration, generate a serial dilution series of 4 to 8 points. It is often good to include a 2x concentration to ensure the antibody is working well. If the optimal concentration is 2X the recommended usage, this can suggest a problem.

Solutions Needed

1. Staining Buffer: Phosphate Buffered Saline with 0.5% Bovine Serum Albumin (Fraction V) (See Leinco Part Number: F1175)
2. Fixation Buffer: 1-5% formaldehyde in PBS (See Leinco Part Number: F1194)
3. Blocking Buffer: 5-10% Normal (species) Serum in Staining Buffer. The species to use is based on the origins of the antibody being used. (Leinco has many Normal Serum species available).

Protocol (using 12x75 mm tubes)

  1. Prepare cells at a final concentration of 2.5×104 per µl in blocking buffer
  2. Aliquot 40 µl of cells per sample
    1. If using a viability dye, include a single stained control for viability (for compensation)
  3. Prepare the dilution series
    1. Prepare the concentration so that 10 µl can be added to each sample
  4. Add the antibody to the cells and gently mix
  5. Place on ice, in the dark, for 20 minutes
  6. Add 3 ml staining buffer to the sample
  7. Centrifuge the sample at 900xg
  8. Aspirate the solution while not disturbing the pellet
  9. Resuspend the sample in ~300 µl final volume
    1. If using viability dye, add to the sample
    2. If sample has to be fixed, resuspend in~300 µl of fixation buffer
      1. If using fixation buffer, cell impermeant viability dyes (PI, 7AAD, DAPI, etc) cannot be used
  10. Analyze the cells on the flow cytometer
    1. Collect sufficient events for analysis (10,000 positive events)
  11. Export the data and analyze in software of choice
  12. Calculate the Staining Index (SI) and plot versus concentration of antibody3
    1. Staining Index (SI) = ((medianpos-medianneg)/((84%neg-medianneg)*0.995)

Typical Results

As is shown in this curve, the titration reveals the optimal concentration (shown in green). As the antibody is increased, the staining index decreases because of the increase in background binding. On the other side, as the concentration decreases, the staining index decreases because the antibody is not at saturating concentrations.

Protocol (using 96 well plates)

  1. Prepare cells at a final concentration of 2.5×104 per µl in blocking buffer
  2. Aliquot 40 µl of cells per sample
    1. If using a viability dye, make sure to include a single stained control for viability (for compensation)
  3. Prepare the dilution series
    1. Prepare the concentration so that 10 µl can be added to each sample
  4. Add the antibody to the cells and gently mix.
  5. Place on ice, in the dark, for 20 minutes
  6. Add ~300 µl staining buffer to each well
  7. Centrifuge the plate at 900xg
  8. Aspirate (or Flick) the solution out of the wells
  9. Repeat steps 6-8 two more times
  10. Resuspend the sample in ~200 µl final volume
    1. If using viability dye, add to the sample
    2. If sample has to be fixed, resuspend in~200 µl of fixation buffer
      1. If using fixation buffer, cell impermeant viability dyes (PI, 7AAD, DAPI, etc) cannot be used
  11. Analyze the cells on the flow cytometer
    1. Collect sufficient events for analysis (10,000 positive events)
  12. Export the data and analyze in software of choice
  13. Calculate the Staining Index (SI) and plot versus concentration of antibody3
    1. Staining Index (SI) = ((medianpos-medianneg)/((84%neg-medianneg)*0.995)

References

1. Hulspas R, O’Gorman MR, Wood BL, Gratama JW, Sutherland DR. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom 2009;76:355-64.
2. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 2006;69:1037-42.
3. Telford WG, Babin SA, Khorev SV, Rowe SH. Green fiber lasers: an alternative to traditional DPSS green lasers for flow cytometry. Cytometry A 2009;75:1031-9.

Special Acknowledgment and Thanks to the Siteman Cancer Flow Cytometry Core Facility Core Director Mr. Bill Eades and The University of Rochester Medical Center Flow Cytometry Shared Resource Share Resource Direct Dr. Timothy Bushnell, Ph D for their review and input on this technical document.

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