RE: Sorry-one more question...and a rant

Hi Janet:
Yes, we have all been there and feel your pain. Here are some things
that I have felt really help:
1) Make sure you have a fluorescent scope in your lab and look at the
samples with the individual. Often I find what they are calling
"brightly shining" is autofluorescence (this can easily be proven with
the appropriate controls).
2) If you can't see it on the sorter, run it on another instrument. If
it can't be seen on either it is not there. 
3) I find you can head off a lot of problems by scheduling a presort
consult with people who you have not sorted for before or are trying a
new protocol.
Here you can make helpful suggestions about the best way to prep samples
and you can check their "experimental design logic" (if there is any).
Having lots of published protocols on hand helps as well. 
4) You can also suggest they bring the appropriate controls (at least
the first time) that would help figure out whether the problem is their
sample (most often) or your instrument (almost never). 
5) I usually suggest they do a pilot run with a small sample to make
sure we can "see" what they want to sort and that their reagents and
staining protocols are working. This can either be run on a benchtop or
the sorter. I motivate them to do this by offering to run the "test"
sample for free, with the understanding that if they do the real thing
on the first sort and it doesn't work they will be charged anyway. You
can only lead a horse to water; you can't force it to drink.

This job requires a lot of patience and a desire to help investigators
do things better (or correctly). There will always be those that no
matter what you do or say will insist on doing things their way and
blame you if it doesn't work. But I have found that those are few and
the majority are just grad students or post docs who are not getting a
great deal of guidance (or are being misguided, sometimes by awful
publications!) or even PIs who are utilizing a technology with which
they do not have a great deal of experience. Keeping a sense of humor
and a gentle manner will help you much in the long run.
Hang in there!!

Joanne Lannigan, MS
Director, Flow Cytometry Core Facility
University of Virginia
Jordan Hall, Room 7067
P.O. Box 800734
Charlottesville, VA 22908-0734
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan@virginia.edu

> -----Original Message-----
> From: janet dow [mailto:jldow@unity.ncsu.edu]
> Sent: Thursday, July 15, 2004 9:16 AM
> To: Cytometry Mailing List
> Subject: Sorry-one more question...and a rant
> 
> Dear Flowers.
> 
> Thank you to all who replied about my question about quantitating GFP
> .  Most replies suggested a bead kit mainly from BD.	Here is the
> info if you are interested.  I will buy the kit and try it.
> 
> You might try the EGFP calibration beads available from BD Clontech .
. .
> cat# 632394.
> 
> 
> I now have another question and a little rant to all who I know will
> appreciate it.	I have had several clients bring me samples
which
> they claim to have examined under fluorescence scope and can see
> their labeled cells shining brightly.  We then run the sample through
> the machine and can see little or no signal.
> 
> Can someone give me a terribly scientific explanation for this that I
> can rattle off to these people who want to blame me when their
> experiment doesn't work???
> 
> Okay here is the rant:
> 
> 
> Also is anyone else tired of being blamed when experiments don't
> work?!?  I get really tired of having to try to help trouble shoot
> experiments for people only to find out 20 minutes into the
> discussion that this experiment has never been tried before or they
> are using an antibody developed for a different species or they are
> using a transvection vector developed for bacteria on yeast cells.
> They just can't understand why it didn't work and it must be
> something that I am doing.   But my favorite is when PIs send
> graduate students or post docs to do all the work, and then contact
> me later because the experiments haven't  worked and are mad because
> they are being charged for it.	Of course, it is all my fault
the
> experiments didn't work and how can I have the nerve to charge them
> for it!!!
> 
> 
> Okay that is enough and I know I am preaching to the choir, so thanks
> for listening.	The truth is I am really luck and my boss
totally
> backs me up and defends me relentlessly to these people.  The really
> fun part is my coworkers in my other lab always know when I have just
> dealt with one of these people as I will immediately go downstairs
> and get chocolate from the special super candy bar fund in our
> basement.
> Thank god for chocolate and peanut butter.
> 
> Again thanks for listening and for being the only ones who can truly
> appreciate.
> 
> Thanks again for all the replies
> 
> Janet Dow
> 
> 
> --
> Janet Dow
> Laboratory Research Specialist and Manager
> Flow Cytometry and Cell Sorting Facility
> North Carolina State College of Veterinary Medicine
> Room C-309
> Raleigh, NC 27606
> (919)513-6443