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V.$LÓPEZ$RODAS$y"col.$

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2.3./Lectin=binding/pattern/

Lectin?binding! was! developed! using! different! eight! FITC?labelled! lectins!
showed! in! Table! 1! (Sigma! Chemical! Company,! USA).! Each! of! the! 35! strains! was!
treated! with! the! lectin! at! a! 0.1! µM! dilution! as! previously! described! (48,! 50! 56).!
Cells! were! centrifugated,! in! order! to! separate! the! supernatant! and! collected! the!
pellet! (2500! x! g,! 20! min),! rinsed! with! BSA! to! prevent! nonspecific! lectin! binding,!
and!aliquots!of!105!±!103!cells!were!incubated!with!each!fluorescent!lectin!for!1!h!
at! 20ºC.! Subsequently,! cells! were! washed! three! times! in! PBS.! Additionally,! water!
samples!from!the!four!different!M.)aeruginosa)blooms!were!treated!with!the!lectins!
employing!the!same!procedure.!

Table$1.?!Lectins!used!in!this!study.! Abbreviation$ Specificity$

Lectin$

Arachis/hypogaea/ PNA! GalNAc$

Canavalia/ensiformis// ConA! Man,$Glc$

Glycine/maxima/ SBA! GalNAc,$Gal$

Phytolacca/americana/ PWM! GlcNAc$

Pisum/sativum/ PEA! Man,$Glc$

Psophocarpus/ PTA! Gal$
tetragonolobus/

Triticum/vulgaris/ WGA! (GlcNAc)1B3$

Ulex/europaeus/I/ UEABI$ Fuc$

!
The!washed!cells!were!evaluated!through!a!Zeiss!Axiovert!microscope!with!

FITC! filter! set! (450?490! nm! excitation! and! 520?560! nm! emission)! for! epi?
fluorescence.!All!tests!were!read!“blind”:!the!person!reading!the!test!results!did!not!
know!the!origin!of!the!tested!material.!Three!different!observers!read!all!samples.!
In! all! cases,! positive! (cells! stained! with! acridine! orange)! and! negative! (cells!
incubated!with!FITC!non?labelled!lectins)!controls!were!used.!

Images!of!lecting!binding!were!captured!using!a!digital!camera!(Canon!EOS!
450d)! connected! to! a! microscopy! and! were! processed! using! an! image! analysis!
programme.!!

In! addition,! Statically! analysis! were! done! by! calculating! a! non! linear!
regression!analysis!between!toxin!production!(in!µg!microcystin!LR?equivalent!per!
cell)!versus!the!number!of!lectin!that!positively!label!the!strain,!using!the!computer!

128$
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