Kinetic studis using flow cytometry

One parameter that isn't used to a great extent in our facility, if at all, is the time parameter. From a diagnostic point of view time is useful to assess flow rate; if we do a dot plot with time as the x-parameter and fluorescence for example on the y-axis we can determine the flow and whether or not it is disrupted as the sample goes through.

We can however use it to assess dynamic changes in samples, for example to assess changes in fluorescence if we use a calcium or other ion indicator.

A number of papers have been published on this. We can use dyes such as Fluo3, which is excited by the blue 488 laser and emits in the green FL1 channel (Calibur). As with many dyes of this sort fluorescence intensity increases upon binding of calcium. Ratiometric measurements require a near-UV laser to excite Indo-1 and so is restricted to instruments with this excitation source. Recently however the green dye referred to above can be used in conjunction with Fura Red to perform ratiometric imaging by collecting green and red fluorescence simultaniosly. With the advent of more collection channels and lasers we can now of course combine calcium measurements with immunophenotyping and viability assays to really unpick the complxities of multicellular systems.

One issue that exists however with performing calcium flux on machines that are presurised (eg FACSCalibur) is that sample tubes have to be physically removed from the intake port, agonist added, then placed back on for further acquisition. This produces a gap in the data. With this gap its hard to determine what has occurred during this initial stage. A system from Accuri appears to have overcome this. As it is not a pressurised system one can add agonists or other stimulants directly into the tube as acquisition occurs negating the need to pause acquisition and preventing the loss of data we see on more conventional systems. Neat.

We have recently been contacted by a researcher interested in multicolour calcium imaging and we are currently planning to conduct a workshop this autumn on the subject. If you would be interested please let me know.