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> What do people do about subtraction of isotype
> control values from flow determination of the so-called "% positive"
> population?
[snip]
> When I follow this through algebraically, I come up with the following
> formula:
>
> y=(x-c)/(1-c/100)
Yes, your equation makes perfect sense for estimating the percentage
of the _originally negative_ cells which were stained positive. The
same equation has been used for >25 years to estimate the fraction
of target cells lysed, where C is the percentage 'spontaneous release'
of 51Cr (where the applicability of the equation is more debatable).
See ref. below*
Changing subject a bit, what bothers me is the occasional use of the cut
line set on the control (e.g. 99% of control below the line) to estimate %
positive when the entire distribution shifts slightly to the right (left
edge as much as right edge) without changing shape. Although 20% may now
be above the cut line, seems to me the result is that approximately 100% of
the cells are (weakly) positive. The 20% is meaningless since the cells
which were dimmest before staining have become just as much brighter as
have the cells which were brightest before staining. Thus, seems to me the
cut line should be used to estimate % positive as <100% only when the shape
of the distribution changes, consistent with bimodality (at least a bump or
tail on the right).
*Martz, E. 1993 The 51Cr-release assay for CTL-mediated target cell lysis.
In: "Cytotoxic cells: recognition, effector function, generation, and
methods", Sitkovsky, M. V.; Henkart, P. A., eds., Birkh!user; Boston MA USA
pp. 457-67. (reprint available on request)
/*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -*/
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