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Cd11b detection

Cd11b detection - Flow Cytometry Forum

Cd11b detection - Flow Cytometry Forum. Discuss FACS cell sorting, flow cytometry, analysis and cell staining.


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  #1  
Old 01-28-2010, 04:25 PM
Pipette Filler
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Default Cd11b detection



Hi,
I’m trying to characterize by FACS the activation of murine neutrophils using different markers among which CD11b. I have some issue with this marker because I obtain a different (time dependent) profile of activation every time that I performed this type of analysis. I hadn’t these problems with CD62L…Can someone give me some suggestions?
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Old 02-01-2010, 12:52 PM
Pipette Filler
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Default Re: Cd11b detection

Hi Ale,

I´m using CD11b on human neutrophils quite often...this is an interesting marker, but can be difficult to obtain comparable results due to very easy activation. I just can give you some basic advice: try to keep PMN on ice where possible. fix your cells with PFA as soon as possible. Take extreme care of good islation procedure. Avoid shaking, fast pipetting etc. Maybe its ok for your setup to compare expression in relation to a control for each experiment; you could then express cummulative results in fold increase over baseline for example.

Cheers

Björn
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Old 02-05-2010, 04:29 PM
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Default Re: Cd11b detection

Hi Björn,
I isolate the PMN from the bone marrow using the MACS system. After the isolation I plate the cells (500 000) in 24 well plates and I treat them for 30’, 1h, 2h, 6h, 12h, 18h and 24h at 37°C. As control I use untreated cells. After the different time points I put the plate in ice and I collect the cells using the pipette flush (the cells are a little bit attached to the plate so it’s necessary to pipette several times…). I collect the 30’, 1h, 2h and 6h all together one day, the day after in the morning the 12h and 18h and in the afternoon the 24h…All the passages after the treatments are in ice and the centrifugation are at 4°C. After the staining I fix the cells…At what point is it better to fix the cells? How can I collect the cells (without pipetting)?
Thank you for your help!

Ale
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Old 02-06-2010, 09:08 AM
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Default Re: Cd11b detection

Hi Ale,

hm, I only used the MACS for a couple of time because I needed more PMN than I could isolate with this system. But according to the literature, your neutrophils should be at a very low activation level after this isolation procedure (after all, there are no long centrifugation steps as in Ficoll-isolation etc. ). This shouldn´t be a problem for you...
But keep in mind that pmn are very delicate cells, and unfortunately, they don´t like long incubation. Take a look at Wolach et al, 2007 (PMID: 17379064), they got only 26% living PMN after 16-18h incubation (w/o growth factors). So I could imagine that at least for your samples older 4-6h, your inconsistent results might be, at least in part, due to unspecific binding of your AB to dead/dying PMN...
I would run an apoptosis assay on all your timepoints, and fix your cells in 2% PFA directly after harvesting (altough you keep them on ice, my experience is that CD11b still changes...). CAVE: at least for Annexin-apoptosis-assays, do not fix these cells! (Annexin sort of tests your pmn membranes integrity, and you will get a lot of false-positives after fixation!)

hope that helps

cheers

björn
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Old 02-22-2012, 08:15 PM
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Default Re: Cd11b detection

thanks
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