Flow cytometric (FCM) measurement of intracellular free calcium [Ca2+]i, transients is usually done by two methods: (a) after a short prerun period to assess the baseline the measurement is stopped, stimulus is added and the measurement continued or (b) stimulus is injected during measurement and the sample pressure briefly increased to deliver cells rapidly to the detection point. In (a) measurement of very short transients [Ca2+]i is impeded by the lag time between stimulus addition and restart of acquisition. In (b) response of pressure sensitive cells is hard to analyze. Furthermore, (a) and (b) do not allow to quantify and sort rare responders. A simple Fixed- Time device has been developed. Ca2+ sensitive fluorescent dye labeled cells and a stimulus are placed in different vials. Both fluids are forced by the same pressure through tubing that merges into a T-junction where they mix and are delivered through a connecting tube to the FCM: [Ca2+]i is measured at a certain time after stimulation that is adjusted by sample flow rate and length of the connecting tube. With Fixed-Time, the pressure sensitive neuronal NH15-CA2 cell was analyzed. Furthermore, rare neurotransmitter responsive fibroblast from normal and transfected cultures were sorted and cloned and their dose response characterized. The results demonstrate that fixed- time FCM is an important tool for the analysis of the cells physiology and the preparation of responders.
Attila Tarnok, Attila Tarnok,
"Applications of fixed-time flow cytometry in cell biology", Proc. SPIE 3260, Optical Investigations of Cells In Vitro and In Vivo, (29 April 1998); doi: 10.1117/12.307101; https://doi.org/10.1117/12.307101