titering antibodies and other basic questions

kennedy.jm.2@pg.com kennedy.jm.2 at pg.com
Wed Mar 13 12:43:51 EST 2002


To All,

I am a beginner Flow user trying to teach myself the basics without much help.
Our lab just obtained a new Coulter Altra Flow Cytometer (somewhat
unexpectedly).  I've taken the basic operator's course provided by Beckman
Coulter so at least I can turn on and align the instrument and I'm getting quite
good at running flow check beads.  However, I'm still very uncertain about
preparing and running cells so I hope some of you will help me with my basic
questions.  Just the other day I obtained a copy of Current Protocols in
Cytometry hoping that would answer my questions, but I still need some help.

1)  I will be running mostly endothelial cells and other established cell lines
such as HEK 293 on our flow, but most of the protocols I come accross are for
blood samples and I sometimes have trouble distinguishing which procedures would
apply to cultured cells also.  For example, in Current Protocols page 4.1.2,
under the titering antibodies protocol, step 5 says add 3ml lysing solution.  Am
I correct in assuming this step is just for blood samples and I should eliminate
it when preparing my endothelial cells?

2)  I am not quite understanding the protcol for titering of indirect antibody
to extracellular antigen (page 4.1.3 CP), specificly how do you titer the
primary ab if you don't know the correct titer of the secondary, and vice versa
how do you titer the secondary if you don't know the correct titer of the
primary?

3)  What is the purpose of adding formaldehyde and fixing the cells before
running on flow?  Some protocols have this step and some don't.  Obviously, if
you were preparing cells to sort and grow after you would not add formaldehyde,
so what are the times when you would want to use it?

4)  Do people routinely add normal IgG (or serum?) of the same species the
secondary ab is made in to their cells before binding to block non-specific
interactions?

Thanks for your help,
Jennifer




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