A Quantitative Method for Comparing the Brightness of Antibody‐dye Reagents and Estimating Antibodies Bound per Cell

Aaron B. Kantor1, Wayne A. Moore1, Stephen Meehan1, David R. Parks1

1 Department of Genetics, Stanford University, California, Stanford
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.30
DOI:  10.1002/cpcy.6
Online Posting Date:  July, 2016
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We present a quantitative method for comparing the brightness of antibody‐dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test‐fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test‐fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.

Keywords: antibody reagents; calibration; flow cytometry; fluorescence

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Table of Contents

  • Introduction
  • Basic Protocol 1: A Method for Calibrating Measurements with Dye‐Antibody Reagents and Comparing the Brightness of Immunofluorescence Dyes on Antibody Capture Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: A Method for Calibrating Measurements with Dye‐Antibody Reagents and Comparing the Brightness of Immunofluorescence Dyes on Antibody Capture Beads

  • Anti‐mouse Ig‐κ microspheres (beads) (BD Biosciences Comp Beads, cat. no. 552843 or similar product)
  • Staining medium (see recipe)
  • Antibody reagents:
  • Test reagents
  • Multiple reagents with different fluorochromes, lots, and manufacturers to be compared: Anti‐human CD8 clones RPA‐T8, Mouse IgG1κ and 3B5, mouse IgG2aκ, were conjugated with various dyes, and used in the work illustrated here (details on vendors and fluorochromes are presented in Table 1.30.1; antibody reagents should be pretitrated before running the complementation protocol
  • Ab‐PE 1:1 reagents:
  • BD Quantibrite CD20‐PE 1:1 (cat. no. 347201, ≥95% 1:1)
  • Alternative Ab‐PE 1:1 reagents (with ratio determined by size exclusion chromatography) yield equivalent results
  • Fill reagent(s):
  • Anti‐CD8‐FITC and anti‐CD8‐APC (Clone RPA‐T8) are used here (the FITC fill reagent is used the most extensively; the APC fill reagent enables evaluation of FITC and AF488 test reagents)
  • Quantibrite PE Beads, lyophilized (BD Biosciences, cat. no. 340495): resuspend according to manufacturer's instructions
  • Rainbow calibration beads (multi‐dye reference particles; Spherotech special lot similar to cat. no. RCP‐30‐5 A‐2, 3.0‐3.4 µm) or similar product.
  • 16% (w/v) Formaldehyde solution (Thermo Scientific, cat no. 28908) or similar product, diluted to 1% to 4%
  • Cytometer setup and tracking beads (CS&T, BD Biosciences, cat. no. 642412) or similar product
  • Microtiter plates, 96‐well, flexible U‐bottom, vinyl, 400 µl, non‐sterile (Fisher Scientific cat. no. 14‐245‐140, Thermo Scientific cat. no. 2101) or similar
  • Assorted calibrated single and multichannel pipets (e.g., 1‐10, 1‐20, 10‐50, 50‐300 μl) with appropriate tips
  • Cytometer: Any cytometer providing good linearity and good measurement sensitivity for the dyes of interest should be suitable for this work (systems with analog log amps are not recommended due to their deviations from linearity; a custom 4‐laser LSRII at the Stanford Shared FACS Facility is used here (https://facs.stanford.edu/lsr2_config); it is monitored with CS&T beads on a daily basis and with rainbow calibration beads during each experiment
  • Software:
  • Diva Data Acquisition (BD Biosciences) software or similar
  • FlowJo (FlowJo, LLC, http://www.flowjo.com/) data analysis software (used here) or a similar product
  • Microsoft Excel 2013 with regression application (used here)
  • JMP (SAS Institute, www.jmp.com) (used here)
  • Or other statistical software that offers quadratic fitting with errors of estimate for the fit parameters
  • Vortex mixer
  • Centrifuge for spinning plates and tubes, with appropriate adaptors
  • Vacuum aspirator with double trap with bleach in first flask, filter to protect vacuum, and fine‐tip Pasteur pipets
  • FACS tubes, 12 × 75 mm (BD Falcon, cat. no. 352008) or equivalent
  • Incubator, humidified, 37°C, 5% CO 2
  • 30‐μm Nylon mesh (e.g., Miltenyi Pre‐Separation Filter, cat. no. 130‐041‐407); fabric store mesh can also be used
  • Cell counter, e.g., fluorescence microscope with a hemacytometer and EB/AO stain, or an automated instrument that provides live and dead counts
Table 1.0.1   MaterialsQuantitative Antibody Brightness (Spe/Ab)a,b

Dye Antigen Source Clone QAb (Spe/Ab)c Spe/Ab (relDye Av)d ABD (units/Ab)e
PE CD20 BD QB 1:1 L27 2.1432 1.21 1.53
PE CD8 BL 1:1 '878 RPA‐T8 1.9221 1.08 1.37
PE CD20 BD QB 1:1 L27 1.9109 1.08 1.36
PE CD8 BL 1:1 '878 RPA‐T8 1.9033 1.07 1.36
PE CD8 BL 1:1 '490 RPA‐T8 1.8896 1.06 1.35
PE CD20 BD QB 1:1 L27 1.8178 1.02 1.30
PE CD8 BD '953 RPA‐T8 1.8066 1.02 1.29
PE CD8 BD '953 RPA‐T8 1.8061 1.02 1.29
PE CD8 BL 1:1 '490 RPA‐T8 1.7557 0.99 1.25
PE CD8 BL 1:1 '815 RPA‐T8 1.7550 0.99 1.25
PE CD8 BL >1 '607 RPA‐T8 1.5163 0.85 1.08
PE CD8 BL >1 '607 RPA‐T8 1.4712 0.83 1.05
PE CD8 LT 3B5 1.4489 0.82 1.03
PE‐CF594 CD8 BD RPA‐T8 0.7318
BV421 CD8 BL RPA‐T8 0.3740
QDot605 CD8 LT 3B5 0.4149 1.13
QDot605 CD8 LT 3B5 0.3212 0.87
PE‐TR CD8 LT 3B5 0.3547
PE‐AF610 CD8 LT 3B5 0.2945
QDot655 CD8 LT 3B5 0.2340
APC CD8 BL RPA‐T8 0.2375 1.13 3.66
APC CD8 BL RPA‐T8 0.2020 0.96 3.11
APC CD8 BL RPA‐T8 0.2006 0.95 3.09
CF568 CD8 IL RPA‐T8 0.2083
CF488 CD8 IL RPA‐T8 0.2023
AF647 CD8 BL 3B5 0.1969
APC‐Cy5.5 CD8 LT 3B5 0.1724
CF660C CD8 IL RPA‐T8 0.1339
FITC CD8 BL RPA‐T8 0.1573 1.23 2.14
FITC CD8 BL RPA‐T8 0.1505 1.17 2.04
FITC CD8 BL RPA‐T8 0.1268 0.99 1.72
FITC CD8 BL RPA‐T8 0.1244 0.97 1.69
FITC CD8 BL RPA‐T8 0.1239 0.97 1.68
FITC CD8 LT 3B5 0.1115 0.87 1.51
FITC CD8 BD RPA‐T8 0.1037 0.81 1.41
CF660R CD8 IL RPA‐T8 0.0911
BV711 CD8 BL RPA‐T8 0.0891
BV650 CD8 BL RPA‐T8 0.0755
BV785 CD8 BL RPA‐T8 0.0707
QDot705 CD8 LT 3B5 0.0748 1.15
QDot705 CD8 LT 3B5 0.0556 0.85
CF405M CD8 IL RPA‐T8 0.0562
V500 CD8 BD RPA‐T8 0.0561 7.96
CF405S CD8 IL RPA‐T8 0.0559  
APC‐AF750 CD8 LT 3B5 0.0492
BD V450 CD8 BD RPA‐T8 0.0434 1.42
BV570 CD8 BL RPA‐T8 0.0432
Pacific Blue CD8 BL RPA‐T8 0.0326 1.02
Pacific Blue CD8 BL RPA‐T8 0.0311 0.98
PerCP‐Cy5.5 CD8 BL RPA‐T8 0.0291 1.01
Pacific Orange CD8 LT 3B5 0.0106 1.14
Pacific Orange CD8 LT 3B5 0.0080 0.86
QDot655 CD8 LT 3B5 0.0008

 hInstrument configuration
 aaViolet: 405 nm, 50 mW, Filter: 450/50: V450, BV421, CF405M, CF405S, Pacific Blue; Filter: 560/40 Pacific Orange, V500; Filter 585/42: BV570; Filter 605/40: QDot605; Filter 660/40: BV650, QD655; Filter 710/50: BV711, Qdot705; Filter 780/60: BV785.
 baBlue 488 nm, 50 mW, Filter 525/50: FITC. CF488; Filter 710/50, PerCP‐Cy5.5.
 caGreen 532 nm, 150 mW, Filter 575/25: PE, CF568; Filter 610/20: PE‐TR, PE‐CF594, PE‐AFAF610.
 daRed 640 nm, 40 mW, Filter 670/30: APC, AF647; Filter 710/50, APC‐Cy5.5, CF660C, CF660R; Filter 780/60: APC‐AF750.
 ebReagents: BL = BioLegend, BD = Becton Dickinson, LT = Life Technologies. IL = in‐lab conjugates. The ratio of PE:Ab reagents, 1:1 or >1 is shown if provided. ‘### refers to individual lots. CF amine reactive dyes were obtained from Biotium, Hayward CA. Clones: RPA‐T8 and L27 are mouse IgG1, κ; 3B5 is a mouse IgG2a κ.
 fcQ Ab = Spe per antibody, is shown for each reagent, where Spe = statistical photoelectron scale. Results are ordered by dye average brightness in terms of Q Ab.
 gdrelDye Av is the relative signal in Spe units compared to the average of all the reagents tested with the particular dye, e.g., PE.
 JeABD: antibody brightness relative to the ABD scale for dyes with available lyophilized capture bead samples rated in ABD units (Hoffman and Wood, ). ABD units for each dye represent an assigned fluorescence capability independent of the exact amount of dye or antibody‐dye conjugation ratio in any particular reagent.
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Supplementary Material