Hi everyone!..
I'm trying of conjugate OMVs (Outer Membrane Vesicles) with FTIC (1mg/mL). I incubated 1h (RT) ratios 1:1 of OMVs (Resuspended in PBS) with FITC. Then, I pelleted to 17000rpm/4°C and discarded the supernant with pipete. I do 3 washes with PBS and finally, I resuspended in 100 uL of PBS. I use this OMVs for phagocytosis assay, but I can't see fluorescence.
Any suggestions?
Thanks
FITC is not very reactive at physiological pH. You don't define the composition of your PBS, but typically this will be around 7.2-7.4. This is not high enough for FITC (i.e. the isothiocyanate derivative of fluorescein) to react. You need to elevate the pH to around 9.5 (e.g. use carbonate buffer). I have no experience with OMV's but if they can withstand this pH you should do much better. Or you could use a much more reactive fluorescein derivative (e.g. fluorescein NHS ester). This is unstable (so use as soon as you have dissolved it in DMSO) but it does react at far lower pH values than FITC.
Hi Nick. I used a Binding buffer for conjugate OMV:FITC. This buffer is Na2CO3 50mM with pH: 9.2. However, finally, I resuspend in PBS pH: 7.4. Do you believe that I must resuspend in PBS pH: 9.5? Could I use the binding buffer for resuspend?...
Okay, I misunderstood your original message, pH 9.2 carbonate should be okay so I'm not sure what the problem is now. The incubation time could be extended to 6 hours or overnight. However, you may wish to consider the possibility that conjugation is okay and some other part of the assay isn't working. When you say there is no fluorescence does this refer to your actual assay? If so, can you directly assess the fluorescence of the conjugate in a plate reader, for example? That should settle the question of whether you have a conjugation problem or not.
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