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We find the DCFH-DA assay works best with these conditions:
DCF-DA Stock solution.
100mM DCFH-DA (molecular probes D-399) in DMSO
To 1ml whole blood add 1ul stock sol, i.e a final concentration of 100uM*.
add slowly (over about 5 secs whilst mixing at 37'C) -you can probably just
throw the whole 1ul in in one go, but you know how it is, when you've got a
system that works you dont try and change it!.
Incubate 15 minutes at 37 'C.
Add the agonist of choice
Apply the lysing protocol as previously mentioned
note: whole blood samples (unlike purified neuts) will show -NO-
fluorescence increase with an agonist such as fmlp - you have to prime the
sample first with somthing like gm-csf for about 40 minutes prior to firing
*for purified cells in for example PBS we use 5uM final conc. and obviously
miss out the lysing step.
.. yes this would work perfectly in the Q-prep device, you would have to
scale the technique up though i.e use 100ul whole blood
I hope this is of some help, please let me know if you want any aditional info,
I have pictures of the scatter distributions and/or listmode files if you
or any others are interested -I would have to mail these on a
person-to-person basis as attachments are (quite rightly) frowned upon!
Regards.
Arnold.
>Arnold:
>Some clarrifications about your lysing method...
>yousaid it can cbe used with DCF - at what point in the assay do you
>so lyse and fix? - what concs of DCFH-DA do you use to compensate for
>the extra cells and proteins in whole blood? any more details if you
>have them...
>also - could this work in coulters q prep device?
>regards
>paul
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