[Histonet] 33D1 DC marker- further info
koellingr <@t> comcast.net
koellingr <@t> comcast.net
Wed Feb 7 09:08:26 CST 2007
Sorry, when talking about the cell-culture, of course I meant in-vitro and not in-vivo.
-------------- Original message --------------
From: koellingr <@t> comcast.net
> I guess my very greatest concern is from your first sentence. You did flow but
> "didn't see anything". I guess I would never even attempt IHC if I didn't see
> target verified by flow for an uncharacterized target. Mouse spleenocytes
> stained with 33D1 do give a one log shift and there are enough of them you can
> see them. This is not a shoulder off of the negative peak. I've seen numbers
> of target sites per cell estimated at 10,000 molecules per cell so this should
> be seen. Assuming you are not using a very strange strain of mouse since
> haplotype restriction has not been fully assessed. As far as the BMDC's plated
> to glass after GMCSF stimulation. Were these verified? In-vivo stimulations
> sometimes do fail with resultant lack of expansion of the targeted population.
> My cell culture experimental stimulations were never 100% successful. No ones
> are due to multiple reasons. After 5 days in GMCSF, were those cells flowed? I
> think originally you said the coverslip staining was
> "weak" and that shouldn't be. Check them by flow first. Bad GMCSF, wrong
> dilution calculation? Your stain setup looks good - it does and should work.
> The markers CD11c and CD205 are fine and MIDC-8 is another. But I guess my
> bottom line first and foremost is I wouldn't waste even another second doing IHC
> until I can stain 33D1 in flow and on those coverslips just to be sure my
> control standards are there and functioning. If you are flowing mouse
> splenocytes, can pick up a dendritic cell population by CD11c or 205, and then
> can't see any 33D1 at all - something is wrong long before IHC problems are
> there. Bad mouse, bad 33D1, bad Alexa 488, bad something but it is a lot easier
> to troubleshoot flow or coverslip staining of stimmed BMDC's and get that
> working than to troubleshoot IHC staining up front.
> -------------- Original message --------------
> From: "Martin S."
> Hi All,
> Some further info;
> The sections were mouse spleen - we did some flow analysis with 33D1 of
> splenocytes but didnt see anything - hard to know if this is because there are
> just so few 33D1+ cells or if theres a problem with the antibody. There are a
> few papers out there with quite a lot of staining with 33D1 in mouse spleen -
> most recently in Science (Dudziak et al Vol 315 Jan 2007). For frozen sections I
> have tried 33D1 + mouse anti-rat IgG2b:Biotin with Streptavidin Alexa488 and
> also 33D1 + rb anti-rat:Biotin with tyramide amplification and Streptavidin
> The coverslip DC's were BMDC's - BM cells were harveted from mice and grown with
> GMCSF for 5 days before transfering cells to PLL coated coverslips. Cells were
> allowed to attach for 2hrs and were then fixed with 4% PFA, 10min.
> Basically we want to look at DC's in the spleen in conjuction with a number of
> other cell markers. We are using CD11c and Dec205 antibodies but wanted an
> additional DC marker.
> Thanks for all your suggestions/comments
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