I recently heard that the FACSAria is not a good instrument for the evaluation of absolute counts using beads because of the construction of the flow cell and dual velocity that exists within the flow cell.
However, since the instrument is being frequently used for absolute counts in published work, I was wondering if anyone has come across any publications stating that the FACSAria is unsuitable for this application?
Ina - I believe the FACSAria should not be used for absolute cell counts in the absence of a volume-calibrating bead. Although some cytometers (to name at least two, the Accuri C6 and the MACSQuant) aspirate samples in a volume-determined way, many, including the Aria, retrieve a sample by pressurizing the headspace above the liquid in the sample tube. Because of uncertainties about how robustness of the pressure seal in any individual sample, and uncertainties about the viscosity of the sample, it's not possible to know how much fluid is collected for a given sample. Therefore, while the relative counts are reliable, the absolute counts are not.
If you're including a calibrating bead in each sample with a known concentration, then you should be able to generate reasonable estimates of total cell counts as part of the sample acquisition.
With the Accuri and Miltenyi cytometers, you can obtain an estimate directly.
When we make count estimates, we typically use a Countess or hemocytometer for total counts and use flow for relative quantitation. I'd also note that none of these methods is rock-solid, and I always view counts with some skepticism.
Best, John
Ina - I believe the FACSAria should not be used for absolute cell counts in the absence of a volume-calibrating bead. Although some cytometers (to name at least two, the Accuri C6 and the MACSQuant) aspirate samples in a volume-determined way, many, including the Aria, retrieve a sample by pressurizing the headspace above the liquid in the sample tube. Because of uncertainties about how robustness of the pressure seal in any individual sample, and uncertainties about the viscosity of the sample, it's not possible to know how much fluid is collected for a given sample. Therefore, while the relative counts are reliable, the absolute counts are not.
If you're including a calibrating bead in each sample with a known concentration, then you should be able to generate reasonable estimates of total cell counts as part of the sample acquisition.
With the Accuri and Miltenyi cytometers, you can obtain an estimate directly.
When we make count estimates, we typically use a Countess or hemocytometer for total counts and use flow for relative quantitation. I'd also note that none of these methods is rock-solid, and I always view counts with some skepticism.
Best, John
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