We use the LIVE/DEAD™ BacLight™ from Invitrogen™ (L7012, ThermoFisher Scientific) to look at the effect of drug compounds against Pseudomonas aeruginosa biofilms, in in vitro static conditions. According to the product literature, 3µL of the dye mixture for each mL (1.5µL of Syto9 and Propidium iodide each) is a good place to start. Using confocal microscopy (CLSM) we discovered that at this amount, there is some bleed through of both channels. There is some spectral overlap, resulting in us having to resort to spectral deduction in CLSM.
I would like to know if users go through the process of optimisation of the stains (ie. varying amounts of Syto 9 and PI). What other conditions do you optimise for? Staining time? The manual also states that phosphate wash buffers are not recommended.
Thank you.
I recommend trying one or two concentrations lower of the offending dye(s), but, yes, you could also try lowering the label time. Both can have an effect to reduce the overall staining.
They dyes are not very phosphate-sensitive (for quenching the dyes), but there is some anecdotal evidence that avoiding phosphates can help get a slightly higher signal.
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