Biology Reference
In-Depth Information
techniques of ampli®cation. With certain levels of antibodies, we were able to
separate histograms of speci®c vs. control antibodies with EAS that could not
be distinguished by standard ampli®cation procedures. Second, to obtain a de-
sired level of channel separation, EAS required 10- to 100-fold less antibody
than standard ampli®cation techniques. With less antibody needed to obtain
channel separation, we postulated that background staining would be mini-
mized. Thus, we were encouraged to speculate that EAS could be used to detect
molecules on the cell surface that had never been detected before.
The original impetus for our investigating enhanced signals in ¯ow cyto-
metry was the inability to detect surface Fas ligand by ¯ow cytometry, although
expression was easily detected in a bioassay ( Kaplan and Smith, 1999). Testing
a variety of transfected cell lines that did not show any speci®c staining for Fas
ligand but that did possess Fas ligand activity, we were able to demonstrate Fas
ligand surface expression by ampli®cation staining (data not shown). To test
our procedure in a rigorous way, we assessed PBMC that had been stimulated
with phytohemagglutinin and interleukin ( IL)-2 for 3 days of culture and then
with phorbol myristic acetate ( PMA) and ionomycin for 1 day. A similar pro-
tocol had been demonstrated to induce Fas ligand activity, but ¯ow cytometric
analysis of these cells did not demonstrate a de®nitive population of positive
cells (Tanaka et al., 1996). Whereas we found that the standard staining tech-
nique did not demonstrate any expression of surface Fas ligand, ampli®cation
staining of these cells showed that 10±15% of the cells were expressing enough
Fas ligand to de®ne a separate positive subpopulation ( Fig. 17.2A). It should
be noted that the ampli®cation method di¨ered from the standard procedure
only by the inclusion of the enzyme-catalyzed reporter deposition steps.
To ascertain that the staining we observed by the ampli®cation procedure
was signi®cant in terms of the function of the cells, we sorted the positive pop-
ulation and the negative population and assessed their cytotoxic potential
against Fas-expressing Jurkat targets (Sieg et al., 1996, 1997; Smith et al., 1998)
(Fig. 17.2B). The results demonstrate that our ampli®cation staining technique
successfully identi®ed functionally signi®cant levels of Fas ligand on the cell
surface. Moreover, they show that cells treated for ampli®cation staining re-
tained their viability and function, and further studies have demonstrated that
the cells can continue to proliferate as well.
We have also used EAS to demonstrate annexin V staining of cells under-
going apoptosis. In this case, the staining was veri®ed with an independent
measure of apoptosis ( Kaplan and Smith, 1999). Thus, with two di¨erent mol-
ecules whose presence could be veri®ed independently by functional attributes,
we have shown that EAS provides stainings that are signi®cant. These data
indicate that the histograms obtained by EAS are reliable and meaningful.
Although in most cases it is di½cult to validate staining because there is no
readily measured independent parameter for most targeted molecules, veri®ca-
tion of the staining of Fas ligand and phosphatidylserine gives us con®dence in
the application of EAS to other molecules.
