FACS Related Problem
kelley@mrc.com
Mon, 27 Sep 93 13:53:19 -0400
In message Fri, 24 Sep 93 20:16:40 +0530, sanjeev@nii.ernet.in writes:
> A certain molecule is expressed on the surface of two different cell lines
> at similar levels, but (probably) in two different conformations. I have a
> monoclonal that (probably!) discriminates between the two conformations. In
> comparing the staining pattern of these two cell lines I face the
> following problem.
> The (no first Ab) conjugate controls for these two cell lines stain at two
> different levels, so that it becomes impossible to compare the test
> stainings.
> 1)If I change the voltage for one cell line to match its control staining
> to that of the other, won't it bring about a more dramatic (and unreal)
> effect on the test staining for this particular cell line?
> 2)Or can I just superimpose the two control stainings and compare then
> compare the stainings with the monoclonal in question?
> If anyone could throw some light on this aspect of FACS data analysis I
> will be really greatful.
Sanjeev,
- From the information you give, it is difficult to make very specific
suggestions. If you are trying to work out whether populations are simply
positive or negative for each sample, and they form a biphasic distribution,
then you have no problem, compare each positive staining with its own
control. If however you are looking at shifts in fluorescence intensity
within a single population in each case, I would suggest that you calculate
the ratio between the median value for labeled and control cells for each
line, and compare those. By the way, are your cell lines of different size,
because that may have an effect on both specific and non-specific
binding. This is probably important in your experiment if you are trying to
sort out the relative expression of two isoforms using a single antibody.
It is difficult to be specific with the limited information you give
Have fun
>Mike Salmon
>Department of Rheumatology, Birmingham University UK.
I would add the following to this discussion:
Are the cells of similar autofluorescence when they are run
totally unstained? If not then you are facing a situation that
would best be approached by Alice Givan's suggestions since you
have no control over the most basic of signals. If the cells show
the same level of autofluorescence
you might consider blocking with an irrelevant protein to normalize
the second antibody controls. If by blocking you are able to get
the second antibody staining equal for both cell lines I feel it would
then be valid to do direct comparisons of the monoclonal staining for
the two lines. Alternatively you could try second antibody from another
source (or species) which might not display the selectivity for one
cell line or the other. An interesting situation, please keep us
posted as to your progress.
Keith
**********************************************************************
*
Keith A. Kelley kelley@mrc.com * A wink is as good
Miles Research Center Ph 203-937-2872 * as a nod to a
400 Morgan Lane Fax 203-937-6923 * blind horse.
West Haven CT 06516 *
*
**********************************************************************
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
