Summary Document

This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.

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Dear Flowers,
Thank you for the many responses I got to my question regarding the
best way to obtain cell counts on a flow cytometer. Here is the
summary of all the responses I got.

Kanika Ghai
Graduate Student
The Ohio State University
4185 Postle Hall,
305 West 12th Ave,
Columbus, Ohio 43210

ORIGINAL QUESTION:
Dear Flowers,
What are the best ways to determine absolute cell counts on a BD
FACSCalibur, without needing to run samples on a hematology analyser?
Has anyone used Trucount by BD? Are there other methods/inexpensive
techniques that you have used successfully? What kind of
beads/microspheres can I use that will be compatible with the
FACSCalibur?
Eagerly awaiting replies..
Thank you!

RESPONSES:
From Delynn Moss :
We have used about all the commercial beads available for absolute
counts on a FACScan. Although BD's TruCount tubes are more
expensive, they appeared to be more consistent, more reliable (even
after expiration date), and more convenient.

From David A. Fahmy
CALTAG Laboratories currently offers the CALTAG
Counting Beads [cat# PCB-100] as an efficient single-platform method for
absolute counts that combines the advantages of direct flow cytometric
immunophenotyping with a double internal standard represented by two
different types of beads. The accuracy of this assay is checked by
verifying that proportion of both types of breads agrees with the
manufacturer's indicated proportion. Attached, in PDF format, I have
included the product's datasheet for further information. Please find
and review this literature. If I could be of any further service to
you, please feel free to contact me. Thank you very much for your
interest in CALTAG Laboratories' products.

From Rudy S. Boleslav
Are you familiar with the Guava PCA-Personal Cell Analyzer? Of the
numerous applications it can do, the most popular is Viacount. This
does absolute cell counting & cell viability assessment with an easy
mix & read procedure. It does not use beads. It is also less
expensive than TrueCount. Let me know if you would like more
information.

From Wayne A. C. Harris
Perfect Count beads (PCB-100) from Caltag are a good alternative.

From William King
You have quite a few choices. I have used Trucount beads on a
Calibur, however, I had several samples whose fluorescense overlapped
the beads fluorescense which invalidates the counting accuracy of the
Trucount beads. I subsequently tried Beckman Coulter's Flow Count
beads and had no issues. They state that there's a 30 day open vial
stability though I have been using some of the bottles for quite some
time without any detectable issues though I store them properly and
minimize thermal cycles. Caltag also makes some absolute counting
beads that I understand have an internal standard for counting
accuracy though I have no personal experience in using them. There
are perhaps other manufacturers.

From Marcelo de Carvalho
Trucount works nicely. I'm currently using this to count absolute
numbers of pDCs and myDCs on whole blood samples (see Vuckovick S et al,
J Immunol Methods. 2004 Jan;284(1-2):73-87 ).
Another useful reference may be : Perruche S et al, J Immunol Methods.
2004 Nov;294(1-2):53-66.

From Phil Barren
Make up a bead standard. (known concentration) and add that to your sample.
I use 10 micron fluorescent beads. I adjust the stock concentration
to 1 E 7 per ml.I then make up my sample in 450 ul and add 50 ul of
beads. So I now have sample that is 500ul (total volume) and contains
500,000 beads or 1 E 6 per ml. I then put the sample on the
instrument and draw a gate around my bead singlet population as
defined by a complex gate. G3 = R1 on scatter and R2 on the bright
FL1 fluorescent population (should be off scale to the right). I then
set my acquisition gate on count for G3 ( R1 and R2). I then count
10,000 events in G3. I then draw a gate around my cells of interest
and ask the question how many cells are in there. For an example if I
counted 10,000 beads and 5,000 cells then my cell concentration in
the sample is one half my bead concentration and is thus 5 E 5 per ml.

From Irina Grigorieva
If you speak of clinical applications, you really have 2 options:
either to use Hematology Analyser (any cell couter will work) or
TrueCount beads. Beads are not that terribly expensive, they work
very well, just follow BD protocols.

From Vijay Nandakumar
You might want to look in to Beckman Coulter Flow count spheres. I
have used them to good measure for my absolute counts.

From Jolene Bradford
Molecular Probes has recently released a counting bead product,
CountBright(tm) absolute counting beads *for flow cytometry* (C36950),
that can be used with the FACSCalibur. I have attached the Product
information sheet for you to look at. Also, Trucount tubes work out well.

From William Godfrey
The standard approach for that kind of cytometer is to put in counting
beads that are calibrated to sample volume. TruCount are a lyophilized
unit-dose version. Bead suspensions are available from a number of
suppliers, including Caltag Counting Beads and Molecular Probes
CountBright beads (disclaimer: shameless Invitrogen plugs). There are
some differences between suppliers in how to gate beads and to assure
precision, but all bead suspensions require good mixing and careful
pipetting.

From Martin Glensbjerg
Have you considered the NucleoCounter SP-100 from ChemoMetec A/S? It
is the most accurate, precise and dedicated system for direct
counting of sperms in a semen sample.

From Denise Teoh
I used Calibrite beads as an indicator of proportion of sample read
but realised
that the beads gave far too variable readouts even in the context of a single
experimental group (e.g. only control samples.. which should have roughly the
same number of cells but ended up seeming to vary by a factor of 10). After
trying all means of optimizing the system, I finally gave up on determining
absolute cell counts and stuck to using % of XYZ cells positive or negative for
ABC as representation of my results.

From Ken McDonald
I've used TruCount and it does work (just follow instructions)
but it can be expensive for you. I've also purchased beads where a known
number of beads per/ml is known and then worked out a flow rate for our
equipment by running 300ul of the bead solution per amount of time. I
did this three times to get an average rate. You then can run an
equivalent volume of your sample for the same time frame and get a
fairly accurate number of cells per time. If you know the initial volume
of your sample you then can calculate the total cells per sample