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Dave,
The cheapest way to do absolute counts is to calibrate instrument flow
rates at each of the flow settings you commonly use for analysis. I think
you'll find flow rate to be very accurate at each setting. The way I do
this is to measure the volume of buffer (measured by weight) aspirated
per unit time. Repeat this measurement w/o cells a minimum of 6-10
times and establish a median flow rate +/- error. Remember to subtract
the initial priming volume used to establish stable flow (the measurement
of this volume can be tricky). Put a stop count on time, I use 5 minutes to
get statistically valid measurements. Then repeat this procedure with
buffer containing cells at the cell concentration you use for analysis an
see if there is an effect on flow rates, there should not be much effect
at [cell] below 10e7 cells/mL.
For your experimental cell counts include fluorescent beads (cheap
ones) to use as a flow stability indicator and monitor their fluorescence
as a function of time. Any break in the fluorescence vs time plot
indicates a flow problem during acquisition. Remember to draw a gate
around the beads and subtract their count from the final total count.
For more details see Rehse, et al Cytometry (Communications in Clinical
Cytometry) 22(4): 317-322.
MarkRehse@CellPro.com
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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