Work InstructionWI0012Rev: A

Determination of Microbead Site Density by Flow Cytometry

  1. Materials
  2. Name: Quantum Simply Cellular Antigen Binding Capacity Beads
  3. Manufacturer: Bangs Laboratories, Inc.
  4. EXAMPLE: Catalog 815 (for use with mouse primary antibodies)
  5. Name: Fluorochrome-Conjugated Primary Antibody to Protein of Interest
  6. EXAMPLE: Anti-P-Selectin: FITC-Conjugated G1
  7. Manufacturer: Ancell
  8. Catalog: 252-040
  9. 500 µg/mL, 200 µL
  10. F:P Ratio: 7.0
  11. Name: Protein-Coated Microbead Sample
  12. EXAMPLE: P-rIgG coated microbeads prepared with WI0006.
  13. Name: Uncoated microbeads (control to match sample)
  14. EXAMPLE: 6 µm Polybeads
  15. Manufacturer: Polysciences, Inc.
  16. Catalog: 07312
  17. 2.1 X 108 particles/mL
  18. EXAMPLE: 10 µm Polybeads
  19. Manufacturer: Polysciences, Inc.
  20. Catalog: 17136
  21. 4.55 X 107 particles/mL
  22. Control Protein
  23. EXAMPLE: Human Serum IgG
  24. Manufacturer: Sigma-Aldrich
  25. Catalog: I2511-10MG
  1. Procedure
  2. Pre-coat 5 microcentrifuge tubes with 0.2µm-filtered 1% tween 20 in PBS for 30 minutes at room temperature.
  3. Prepare negative control microbeads of the same size as the sample of interest.
  4. Coat with the negative control protein using the same protocol used to coat the sample microbeads. Use the same manufactuerer’s lot as the sample. We have evidence of dramatic lot-to-lot variability in the autofluoresence of some microbead products.
  5. Adding 1% tween in PBS during spin steps may help improve recovery for the beads.
  6. Aliquot out samples for adsorption:
  7. Microbead sample of interest: roughly 4 X 105 beads. Dilute in PBS to 100 µL total volume.
  8. Control, non-specific protein (negative control): roughly 4 X 105 beads.
  9. Mix together Antigen Binding Capacity beads:
  10. Use 50µL of each control sample with antigen binding capacity (#1, #2, #3, #4). This will yield 4 X 105 beads in 200 µL.
  11. Spin the ABC beads at 3,000 xg for 4 minutes and resuspend in 100 µL PBS.
  12. Incubate the fluorescently-labeled primary antibody with these 3 samples:
  13. Incubate each of the 3 samples at 10 µg/mL (theoretically 0.4 µg/mL would be required to saturate the high binding capacity microbeads, #4, at 4X106 beads/mL). For a 500 µg/mL antibody stock, use 2 µL stock solutionper sample.
  14. Incubate on rotator for 30 minutes at room temperature.
  15. Add 400 µL of 1% tween in PBS to facilitate the spin down. Rinse twice in 1% tween to remove unbound antibody and resuspend in 400 µL PBS at the end.
  16. Prepare blank controls (to assess autofluorescence):
  17. Use microbeads from the same lot as the samples, roughly 4 X 105 beads. Dilute to 400 µL in PBS.
  18. For Polysciences 6 µm Polybeads this will be 1.9 µL stock in 400 µL PBS.
  19. For Polysciences 10 µm Polybeads this will be 8.8 µL stock in 400 µL PBS.
  20. Prepare the Simply Cellular “B” Sample, one 50 µL aliquot = 1 X 105 beads (add 350 µL PBS) is sufficient.
  21. Analyze all samples with flow cytometry.
  22. Set the fluorescent channel voltage based on the blank and ABC sample and keep this constant for all remaining samples. You may wish to readjust the forward/side-scatter settings for each bead size.
  23. Measure the mixed ABC samples (#1, #2, #3, and #4) and the ABC blank sample. From the Bang’s Laboratory protocol:“Analyze the [ABC] microspheres on the flow cytometer. [ABC] bead populations may be combined or run separately. A flow rate of 100-200 events per second is recommended. Typically, 1000 events are collected per bead population (i.e., 5 bead populations x 1000 events each).”
  24. For the sample blank, negative control, and sample populations, it is recommended to sample 10,000 events each.
  25. Be sure to save the resulting data files for future reference.
  26. Analyze the data to determine the site density.
  27. Open the files in flow cytometry software such as WinMDI. WinMDI is available for free at the Flow Cytometry Core Facility web page (see references). Note that if the file is output from a Mac and you are performing the analysis on a PC, you will need to manually add the .FCS extension for the software to open the file.
  28. Measure the fluorescence of the ABC standards to calculate the sensitivity:
  29. Plot forward scatter vs. fluorescent intensity and gate each subpopulation of the blank and ABC beads. Record the geometric mean fluorescent intensity of each gated population.
  30. Plot the geometric mean intensity of each ABC bead population as a function of the ABC. The ABC value should be provided on each bottle (#1, #2, #3, #4). Subtract off the geometric mean intensity of the blank ABC beads from all datapoints. Fit a line through the results with a forced zero intercept. The results should be highly linear (R2 > .99).
  31. Measure the fluorescence of the blank, negative control, and sample bead populations.
  32. Gate each dataset on a plot of forward scatter versus fluorescent intensity to remove doublets.
  33. Record the geometric mean for each gated dataset.
  34. Subtract the geometric mean from the negative control microbeads from the geometric mean of the sample.
  35. Dividethe control-corrected geometric mean by the sensitivity (slope) calculated from the antigen binding capacity standards to measure the number of antibody molecules bound specifically to the target protein per microbead.
  36. The site density calculation requires an assumption of the protein:antibody ratio, which will vary between 1 and 2.
  37. For optimal results you may wish to repeat the measurement in triplicate.
  1. References
  2. Special Issue: Quantitative Fluorescence Cytometry: An Emerging Consensus. Issue Edited by R. Lenkei, F. Mandy, G. Marti, R. Vogt.Cytometry1998, 33, Issue 2.
  3. Flow Cytometry Core Facility web page: cytometry/
  4. Kerker, M.; Van Dilla, M. A.; Brunsting, A.; Kratohvil, J. P.; Hsu, P.; Wang, D. S.; Gray, J. W.; Langlois, R. G. Is the central dogma of flow cytometry true: that fluorescence intensity is proportional to cellular dye content?Cytometry1982, 3, 71-78.
  5. Product Data Sheet 815. Bang’s Laboratories, Inc.
  1. Revision History

DateRevDescriptionRevision Author

10/04/07ARevised description of measurementBrian J. Schmidt

method. Added additional reference

material.

9/04/07-Initial release.Brian J. Schmidt

bme.virginia.edu/lawrence Date: 10/04/071/3