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Phagoyctosis assay and fluorescent beads - (Oct/15/2008 )

Hi,

I need to realise a phagocytosis assay for macrophage with fluorescent beads (by cytometry). Does anyone of you had any background in this kind of experience. Which beads choose and where can I find it.

Can you help me?

Many Thanks

biofred

-Biofred-

QUOTE (Biofred @ Oct 15 2008, 02:59 PM)
Hi,

I need to realise a phagocytosis assay for macrophage with fluorescent beads (by cytometry). Does anyone of you had any background in this kind of experience. Which beads choose and where can I find it.

Can you help me?

Many Thanks

biofred



Hi,

I have used FluoSpheres Fluorescent Microspheres from Molecular Probes (Invitrogen) to assess phagocytosis by monocytes and macrophages by flow cytometry. You can get them with different fluorescent conjugates. The size that I used was 1.0┬Ám. It might be a good idea to try a red colour because I have found that my macrophages autofluoresce into the green channel (FL1).

See Butler et al (2007) Inflammation Research (56) 353-361 for a reference.

Hope this helps!

-Cork-

Hi Cork, many tanks for those informations. It's clear and concise!

Have a nice day

biofred

-Biofred-

Bangs laboratories sells multiple variations of beads - and MUCH cheaper then most other companies.
I used 0.5-1um beads, PVC with a GFP tag. I noticed almost no background fluorescence and ti was very easy to distinguish.
You can even tell (kind of) the difference between cells that have uptaken 1, 2, 3 beads etc.
It worked damn well.

I also recently used pHRodo labelled staphylococcus aureus particles (invitrogen cells the dye, and pre-labelled particles) for looking at uptake in RAWs and it is utterly amazing. The dye only fluoresces once it is internalised (and the lysosome triggers a pH change)... fluoresces in the red channel (PE stain) and absolutely no background. Results differed to the GFP bead work - since the bead method also shows particles bound to the outside of the cells (despite several washes).

anyway, goodluck - any other questions drop me a line.

-Aaron I-