[Cytometry] Teaching Flow Cytometry

Robert Leif rleif at rleif.com
Sun May 8 13:01:31 EDT 2011


Cytometry Distribution
I may have missed something. However one should mention  the obvious fact
that it often pays to look with a microscope at a flow cytometry
preparation. The old maxim "garbage in = garbage out" still holds. One other
check, particularly in the case of cell dissociation procedures, is to
measure total DNA. It is improper to claim that a cytometry measurement is
representative if only a small percentage of the DNA is recovered.

Parenthetically, did anyone else teach a course in Cytometry prior to
1969-1970?
Yours,
Robert (Bob) C. Leif, Ph.D.
Vice President R&D
Newport Instruments
3345 Hopi Place
SAN DIEGO,CA 92117
Email: rleif at rleif.com

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Elizabeth R. Simons
Sent: Sunday, May 08, 2011 8:12 AM
To: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Teaching Flow Cytometry

As one who has been using flow cytometry for years, let me add to what all
of you have written:
Not only should any course or user indoctrination cover the cellular
properties (and their changes) that flow cytometry can measure and the
enormous amount of information that can be gotten, it should also cover what
it can't do, the assumptions that are involved, the approximations that are
made (e.g. in compensation), precautions that can be taken (narrow band pass
filters vs greater intensity.
I tried to at least introduce these topics when giving talks to potential
users of our core, before then going into more detail on the probes that are
available (or can be created).
It also helps to introduce the concept of time,i.e. the enormous potential
of measuring and corerelating - as only the flow cytometer or very fast
single cell imaging can do - the cell's response with the amount and nature
of a specific stimulus, the cell receptor involved, the change in specific
compartment(s) within the cell, whether a there are responding
sub-populations and what distinguishes them from non-responding ones, etc.
One can, if desired, follow these changes in real time while stirring and
keeping temperatures constant, watch the results after the changes have
reached equilibrium, or sort the cells for any parameter one wishes.
I've found that presenting these possible uses of the instrument leads some
of the listeners to think a bit outside the box of the better known
applications and leads to some terrific new information.
Liz

Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
72 E. Concord Street
Boston, MA 02118
esimons at bu.edu
617-638-4332
Quoting "Nebe-Von-Caron, G" <g.nebe-von-caron at alere.com>:

> Hi Till
>
> First of all you should let  your users appreciate the difference 
> between a flow cytometer and a fax machine, particularly as you use a 
> particle analyser that can do other things than only fluorescent 
> activated cell sorting .  Reading TFM, The Fantastic Manual, is a good 
> start but usually not as entertaining as reading Howards book to learn 
> how to obtain low noise valves for building excellent amplifiers.
> However there are a couple of small "Introduction to Flow Cytometry"
> booklets available for free from vendors who want your address details 
> in return, even Howards big book. Once potential users have read 
> through one of the smaller booklets you have a good discussion basis, 
> so let them read one of those before they come to see the person who 
> is responsible for the cytometer. And learning works much better if 
> you have some basic information about the things you want to learn 
> more about.
>
> You can teach students all sorts of tricks on how to do things and the 
> basic principles of flow cytometry, but the thing that helps to be a 
> really successful cytometrist is to have "the Knack"
> http://www.youtube.com/watch?v=CmYDgncMhXw  The most important element 
> in teaching is to live by example and to transfer the enthusiasm and 
> passion you have for your scicence.  To prime the enthusiasm of others 
> it helps to explain why this technology is such a central research 
> tool in science. It comes from its ability to differentiate cells / 
> particles and generate population statistics as it can measure the 
> relevant numbers and features of cells particles, identify 
> subpopulations/subcommunities with similar properties, find (rare) 
> events and separate events of interest by gating or regions of 
> interest and sometimes even physically separating them. The key is in 
> the appreciation of learning that all systems are made up from 
> individual units with differences from each other as those differences 
> create complexity and stability. To understand the the "big picture" 
> you have to appreciate the differences at the elementary level, or as 
> said by
> Goethe:
> ''to find you in infinity of space and time one must first divide and 
> then combine", the basic theorem of scientific studies such as systems 
> biology.
>
>
> There are a lot of cytometry courses around and usually if you have a 
> manager above you he/she will be much happier to send staff on a 
> course elsewhere than having you set up an in house course. First of 
> all it is difficult to budget, but more important, for a manager a 
> trainer / consultants outside the managers company is always better / 
> more intelligent than in house staff as they are not working under the 
> managers supervision, e.g. some managers tend to have difficulties 
> admitting that someone below them can train people in something that 
> they cannot train in themselves, thus admitting that they are not on 
> top of everything. If your boss is a leader and not a manager that's 
> different. Institutes and Universities are a bit more open in this as 
> they are more used to the idea of teaching but as you have a couple of 
> enthusiasts up / down the road and considering that you are not the 
> only cytometrist in Darmstadt (in fact if I am not mistaken you have 
> even one of the original cytometry specialists living quite near) I 
> would contact some of the local flower gurus for example in Heidelberg 
> as it is always easier to organise training course together with some 
> other cytometrists, particularly if you are not an expert yourself. 
> They tend to be very helpful bunch, each of them having their own area 
> of expertise which they can be very enthusiastic about. Also some of 
> them already run courses, most of them on a break even budget / cost 
> basis and you might as well want to point your new starters to such 
> courses because of the networking opportunity. Other considerations 
> should be whether you can or cannot get an in house budget for the 
> time you spent in training people. If in house training is seen to be 
> "for free" it is usually not treated with the same respect. In the end 
> you still need to conduct some in house training including other steps 
> to consider such as lab internal processes, data handling and 
> particularly health and safety issues. Howard correctly said that 
> unfortunately a lot of students come up with the idea of it all being 
> hush hush wizardry of which they do not need to understand the minor 
> details as they use a cytometer with an attached operator. Ideally 
> they want to be served with a 12 colour antibody panel and someone to 
> prepare things for them and clear up after them, push a button and 
> become a manager writing in their CV that they done 12 colour 
> cytometry. You don't do them a favour by not letting them work out 
> things for themselves as scientific thinking is not achieved by giving 
> them answers but by learning how to phrase the question and what to use as
control.
>
>
>
> Have fun
>
> Gerhard
>
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Howard 
> Shapiro
> Sent: 06 May 2011 14:53
> To: Dettmering, Till
> Cc: hsfc at elist.tufts.edu; flow list Purdue flow list Purdue
> (cytometry at lists.purdue.edu)
> Subject: Re: [Cytometry] Teaching Flow Cytometry
>
> Till Dettmering wrote:
>
>> I'm a PhD student for a year now and I'm working with flow cytometry
> for 2.5 years. Since there is no real dedicated FACS-'guru' in our 
> group who I could have learned from, I had to teach practically 
> everything about flow cytometry to myself, including maintenance of 
> the device (a Partec PAS III). I find myself more often in the 
> situation in which I have to teach new students who have never seen a 
> flow cytometer how to work with it and how to interpret the data. 
> Although it works out quite well, I'm very interested on your 
> experiences on how to best teach new people flow cytometry. What do 
> you start with? Do you have something like curriculum you follow when 
> teaching it? What did you start with when you learned it? What should 
> one pay special attention to when teaching?
>>
>> I'm very interested in hearing your experiences!
>>
>
> Problem number one these days seems to be motivating the new people to 
> think; too many of them want to mix samples with magic juice, put them 
> in the magic box, press the magic buttons on the box and do the 
> necessary magic mouse clicks, keystrokes, etc. on the computer, and 
> save the PowerPoint presentation and the manuscript that will be 
> submitted online to one prestigious journal or another. Then it's back 
> to social media.
>
> Those of us who write flow books and vent on this Mailing List have 
> tried to spell out the basics. Cytometry, as I have often said, begins 
> with the cell, even at the level of the word itself. You have to know 
> what information you want from the cells in your samples, and what 
> reagents you need to use and parameters you need to measure to get 
> that information, and it helps to consider alternatives when they are
there.
>
> The cell identification game is like "Where's Waldo (his name is Wally 
> in some places)?" - The game wouldn't be any fun if Waldo were the 
> only person in the crowd wearing magenta and chartreuse; the 
> intellectual challenge comes in picking out physical features and the 
> patterns of the colors on his clothing and distinguishing them from 
> those on everybody else in the crowd among which he is hidden. 
> Generally speaking, one can always identify a cell from a given 
> species by detecting a specific nucleic acid sequence, or a 
> combination of antigens. In unlysed blood,
> CD45 is good for discriminating white blood cells from red cells but, 
> in cases where you know you won't encounter nucleated red cells, it's 
> much simpler to use a permeant DNA dye. Simpler is always better.
>
> One can learn these kinds of tricks from books and material available 
> online. It's harder to learn how to set up an instrument, verify that 
> it is working properly, and deal with it if it isn't. The theoretical 
> part of that is accessible in books, but which knobs you turn and/or 
> how you set up the software depend entirely on the specific cytometer 
> you're using. Manufacturers offer training courses, and provide 
> manuals, but, as is the case with computers, you can sometimes RTFM 
> and find that it doesn't really tell you all you'd need to set up a 
> flow lab in a postapocalyptic venue. Oral tradition only goes so far; 
> even prehistoric hunters seem to have needed seminars with 
> presentations painted on cave walls.,
>
> When TFM leaves you in the dark, you can get computer questions 
> answered online, sometimes for nothing, or you can buy something like 
> the books in David Pogue's "the missing manual(R)" series, published by
O'Reilly.
>
> What we need in cytometry are equivalents. Practical Flow Cytometry 
> had its genesis in my "Building and Using Flow Cytometers: The 
> Cytomutt Breeder's and Trainer's Manual," and the material from that 
> on building and using instruments, which appeared in the first couple 
> of editions of PFC, has apparently been sufficient to enable dozens of 
> people to put together instruments and keep them running for decades 
> without access to service from outside.
>
> It does not strike me as unreasonable that manufacturers should 
> support their user groups in generating similar documentation, but the 
> users could get organized to do this without the manufacturers' help, 
> which would minimize the likelihood of problems being swept under the 
> rug. The material, which should cover software from both manufacturers 
> and third parties as well as apparatus, should be kept online in a 
> presumably neutral site such as this one.
>
> I've seen people with little or no lab experience get up to speed, at 
> least with relatively simple flow cytometers, in weeks, and people 
> with decades of experience fail to do so in months to years, and I 
> can't always explain either.  There hasn't been a Mozart in cytometry 
> yet, and even he started small. If you can't hear a sonata playing in 
> your head, don't try for a symphony, and, if you can't do two-color 
> fluorescence and two-angle scatter, don't try running a 4-way sorter 
> with 7 beams and
> 24 colors. I spent several years training in surgery, and would have 
> had to spend several more before anybody let me do any significant 
> part of a heart operation; it is, admittedly, harder to kill people by 
> running a flow cytometer incompetently than it is to do so when you 
> are waving a scalpel around vital organs, but the malpractice of 
> adding junk to the medical and scientific literature has its own adverse
consequences.
>
> So let's get organized.
>
> I'll be at my 50th College Reunion during ISAC, which declined my 
> offer to give a plenary talk by remote video, but catch up to me 
> online if you're willing to help.
>
> -Howard
>
>
>
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Elizabeth R. Simons, Ph.D.
Professor of Biochemistry
Boston University School of Medicine
72 E. Concord Street, K407
Boston, MA 02118
617-638-4332 phone
617-638-5339 FAX
esimons at bu.edu
ersimons at earthlink.net


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