Is hemocytometer a reliable way to measure the number of microspheres in a solution? I heard flow cytometers provide the best option but they can only count fluorescent microspheres and not ordinary PS microspheres?
Please suggest the best method for the same?
Rohan, Hemocytometers have long been considered the gold standard for counting cells. Their biggest drawback is that folks don't take the time to learn how to properly load the slide and count. Make sure you read the insert info that comes with the hemocytometer. The ability to count on a flow cytometer depends on which cytometer you have and the size of the spheres - not on the fluorescent signal. For the instrument, you need something like an Accuri or Quant that will allow the user to define a known sample volume from which to acquire events. As for size, very small spheres,
Dear Rohan,
with flow cytometers it can be difficult to measure the count of polystyrene beads, especially if you have a high concentration in your sample and aggregates or doublets are formed, if you gate these out you will get an underestimation of the actual concentration of beads. Also sometimes electronical noise can be a problem if you are measuring small beads, since then the beads might overlap with the noise and this leads to an overestimation of the concentration.
What worked best for me was the use of a machine called the Nanosight (Nanoparticle Tracking Analysis, NTA), which measures the size of the beads using brownian motion, and also measures the conentration. Maybe this machine is also availbale at your institute?
Kind regards, Selma van Staveren
Hi Selma, Thank you for your detailed answer.
Unfortunately we do not have this machine. Also, we are going to measure the beads of size more than 2000 nm, and I don't think the machine could measure that.
I would try to look into the issues mentioned by you and discuss with the technical staff here, to get the possible result.
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