This is in response to Dave Novo's request for less expensive ways to measure absolute counts. 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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