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.
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!
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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
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
