I am currently working on the analysis of human cytokines using the BioLegend LEGENDplex assay on a BD FACSymphony A3. For PMT voltage setup on FSC and SSC, I followed the manufacturer’s protocol using setup beads. However, I encountered some difficulties. At FSC 450 and SSC 300, no distinct bead population appeared on the dot plot. When I increased FSC to 800 and SSC to 600, I could visualize some events clustered near the origin (close to the X–Y axis intersection), but they did not resemble the clearly separated bead populations (groups A and B) shown in the protocol. At this stage, I am unable to resolve the bead populations appropriately. Could you kindly advise on how to optimize the PMT voltage settings or other parameters to better distinguish these bead subsets?
Hi Chit Care
I use a different machine but maybe the following suggestions might help you troubleshoot since the principles are basically the same:
1. Make sure you thoroughly mix the beads before use. Some protocols recommend vortexing for a few mins. Beads tend to settle at the bottom.
2. Run a back flush on your sample line just to make sure you remove any potential clogs. Also, it's recommended to run your beads on "low" flow rate setting, at least until you identify your population. On my machine, that's about 30 events/min.
3. Try scaling your SSC v FSC on Log scale rather than linear using your default voltage first and then adjust accordingly until you find your population.
4. I would also recommend that you run a control with known FSC v SSC to make sure it's not an instrument issue
All the Best!
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