Biology Reference
In-Depth Information
not bind any and is used as an internal negative reference point to set the neg-
ative gate. After adding a saturating amount of the antibody to the beads and
processing them identically to the samples being quanti®ed, the MFI of each
positive bead population is then linearly regressed against antibody binding
capacities of the stained microbeads. The MFI of each antigen being analyzed
on the sample cells can then be converted to ABS by comparison with the
standard curve generated. In contrast to the QSC system, the QB system does
not require prestaining because the beads provided come with a ®xed number of
PE molecules on each population. However, only PE-conjugated antibodies
may be used with this system.
Although the general principles of QFACS are quite simple, meaningful
quanti®cation of ABS requires taking into account the many variables that may
a¨ect the validity of the regression curve generated and the relationship be-
tween the MFI of the sample antigen and the values on the regression curve.
Information provided with each system indicates how to evaluate the standard
curve to determine if staining and instrument conditions were adequate. A
number of the more important variables are discussed below.
Fluorophores
PE has the highest Stokes shift and quantum e½ciency among the common
¯uorophores used in FACS analysis and thus is ``brighter'' than the other ¯uo-
rophores and can lead to higher sensitivities. More importantly, PE does not
self-quench and thus can ensure the linearity of the regression curve at high
densities, making it possible to accurately measure high-density cellular anti-
gens. By contrast, dyes such as ¯uorescein isothiocyanate ( FITC) can self-
quench at higher densities (Haugland, 1996), presumably as a result of their
greater overlap in excitation and emission spectra. Nontandem ``third'' color
dyes such as PerCP, although not subject to self-quenching, are photolabile and
degradation of the ¯uorophore while transiting through the laser beam of the
cytometer makes careful quantitation di½cult at best. Tandem third-color dyes
such as Tricolor
TM
or Cychrome
TM
(PE-cy5) may be a¨ected by inappropriate
intermolecular ¯uorescence energy transfers at high antigen densities (intra-
molecular PE to Cy5 energy transfers are the only desirable ones), once again
complicating the linear response required for accurate quanti®cation. In addi-
tion, especially when using the QB system, it is essential that the ¯uorophore/
antibody conjugate ratio be uniformly close to 1. Because of the large size of PE
(240 kD), chemical conjugation to PE followed by high-performance liquid
chromatography ( HPLC) puri®cation can generally lead to 100% pure 1:1
antibody/PE conjugates (this is also true for protein-chromophores like allo-
phycocyanin). Conjugation to small molecular dyes such as FITC cannot be
controlled to achieve such uniform labeling. Therefore, although standardized
beads exist conjugated to many ¯uorophores, accurate quantitation of ABS
using QFACS requires the use of PE (or other members of its class, e.g. allo-
phycocyanin) for the reasons discussed. Finally, although the newer dyes such
