RE: [Histonet] 33D1 DC marker- further info

Sorry, when talking about the cell-culture, of course I meant in-vitro and not in-vivo.

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> Sonya, 
> 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. 
> Ray 
> -------------- 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 
> Alexa488. 
> 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 
> Sonya 
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