URGENT-CellTrace FarRed DDAO-SE staining protocol

Joost Schuitemaker j.schuitemaker at iqproducts.nl
Thu Jul 14 11:41:31 EST 2005



Hi Lidija,

I worked a lot with DDAOse and I don't understand your mail. No, the 'kit'
doesn't contain a protocol, but it also doesn't contain IgG or
nucleotides. For the protocol part: it depends on the type of cells you
would like to stain and how long you will follow them in time. For human
PBMC 1 uM is sufficient to track the cells after 5 days in culture and
full divisions. Undividing cells can be tracked over a longer period (but
the same concentration). Just stain as you would do with a regular CFDAse
protocol.
- wash cells in PBS
- dilute DDAOse to a concentration of 1 uM, take 1 ml for 10e7 PBMC
- loosen pellet by flicking tube gently and add DDAO solution, mix and
incubate at RT in the dark for 15 minutes. Make sure all cells are in
suspension.
- wash cells twice with PBS/10% FBS (or BSA)
- count and use.
One ampul can be diluted with 200 ul 'dry' DMSO to get a stock solution. I
don't remember the concentration so please recalculate how much you have
to dilute this stock. You stock can be re-frozen for at least 5 times.

Success,

Joost

> I have made a terrible blunder... I have set up an experiment for
> Thursday for which I need to stain cells with DDAO-SE (Molecular Probes'
> CellTrace Far Red). I received the kit last week, but didn't bother to
> check the product insert for the protocol until today. To my horror I
> found that there is no protocol for staining live cells, only IgGs and
> oligonucleotides.
> I have contacted the Probes/Invitrogen people but not being aware of
> their response timek, I thought I would turn to you knowledgeable bunch
> as well.
> I'd be very greatful, if anyone would send me their working protocol for
> staining live cells with DDAO-SE.
> Thanks in advance,
>
> --
> Lidija Bosnjak
> Centre for Virus Research
> Westmead Millennium Institute
> Darcy Rd
> Westmead, NSW 2145
> Australia
>
> Ph: 61 2 9845 9118/9113
> Fax: 61 2 9845 9103
>
>





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