I am wanting to use flow as a diagnostic tool for live/dead sperm counts on an insect that has very tiny, long and featureless non-motile spermatozoa. I am using PI and SYTO16 fluorescent dyes to mark them, but I haven't nailed down a good, consistent protocol yet for the killed sperm control. These sperm are impossible to count or measure using light microscopy. The tails are so fragile and the sperm bundles are difficult to separate. I have followed several published protocols on different invertebrate sperm, but with little success. Our resident flow technician only works with mammalian cells and is unable to help. Any guidance would be greatly appreciated here:)
what size are the sperm? You can purchase sizing beads from Thermo Fisher that range from 200nm to 2um, they also fluoresce and can help you identify the sperm. How many sperm do you have in your sample?
Hi Ann, thanks for your response. I am aware of the beads for counting, but as of yet, no one (myself included) has established any measurement parameters for my beetle's sperm so we could choose the appropriate bead size. We did manage a TEM image of the "head", which is about 150 nm long, but the tails are problematic to measure. They are very long and fragile, and usually break during sample prep. Also, I am using SYTO-17 for live cell stain, but getting very weak fluorescence the way I am preparing my sperm. I was following protocols for SYBR-14. Has anyone substituted SYTO-17 for the green dye in a live/dead assay?
Ok: if the tails are long you should see a good signal on the FSC because the tails pass through the flow cell along their axis, i.e their length. If you plot FSC-A against the green Syto-16 you should be able to find them on the flow cytometer. Also plot FSC-H against Syto-16. You might see the signal piling up on the edge on the right side of the dot plot. You will then need to adjust voltage settings.
The Syto-17 is not good for flow since the excitation and emission are sub-optimal for the red laser, hence fluorescence will look weak. What flow cytometer do you have?
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