Hi, most likely is that you have RBC contamination from your Cord, this has occurred occasionally with us and we have seen the discription of autofluorescence you have observed. This is easily solved by doing repeated wash, centrifugation steps after the isolation of cells from the cord. 

Hope its of use.)

Thanks for the response, but these were cultured HUVEC cell lines purchased from ATCC at passage 2.

Hi, ah ok, we harvest our own, fairly easy procedure as long as you have access to umbilical cord.

This maybe an infection and it maybe you DAPI in the mounting solution that is staining the bugs and DAPI is known to bleed over into all your other wavelengths. Try a mounting solution with out DAPI see if you still get them.

I placed HUVECs from a pellet of 1% PFA fixed cells onto a slide without any dapi or mounting, just PBS. And I still have autofluorescent vesicles.

Hi, How do you make up your PFA? We make 4% PFA by warming up, say 100ml Dh20 to 60oC and add our 4g of Sigma PFA then add 1M NaOH dropwise until remaining PFA powder has dissolved, we freeze this at -20oC as our stock. Our working solution is diluted 1:1 with PBS giving us a final solution of 2% PFA, works perfectley every time. I am not sure why you are fixing your cells in the pellet form. We grow our HUVECs on coverslips and then fix them, also make sure no media or serum is around during fixing as this can use up the fixative effect on the serum rather then your cells, hense also increasing background fluorescence?

Do you have facilities were you can observe fluorescence on live cells, just to eliminate anything auto-fluorescing in your culture?

I wish it were that easy. I just moved labs and I had a group grow the cells and ship to me, including PANC1, H82, MB231 and HUVEC.  They were grown, pelleted and fixed in 1% PFA. I have done this a hundred times, but this is the first time with HUVECs. None of the other cell lines have this. Everything was done at the same time with the same reagents. Any other ideas?

Well as sherlock homes put it,

“When you have eliminated the impossible, whatever remains, however improbable, must be the truth.”

As all the other cells don't have this problem and the only problem is in the HUVECs, it would appear your HUVECs have been contaminated with a auto fluorescing agent. What that agent is remains a mystery, i can assure you HUVECs that we generate do not have this problem. In these circumstances, if it is possible i would repeat the samples.

Endothelial cell derived microparticles are another possibility. You can try to pellet these and try to characterize. Microparticle generation is stimulated under many conditions including altered osmolarity, high glucose, oxidative stress or inflammation.

Hi Subhadeep, do you know if these EC derived particles can autofluoresce if they are fixed with formaldehyde?

Most often EMPs are ectosomes, meaning they are rather budding from the cell surface rather than going-in. (MP/MV shedding is often a way to get rid of unwanted material e.g. Caspase 3 for dying cells trying to rescue themselves).

Did you think about Weibel-Palade bodies ? (although I do not remember whether these are autofluoerscent, they are sub-cellular bodies surely specific for ECs, including HUVECs). You could try to stain with anti-VWF antibodies if you want to check. See George F et al Throm Haemost 1992 67 (1) 147-153.

Hi all. My colleagues usually work with EMP from either HUVEC or other types of endothelial cells and I never heard them speaking about autofluorescent microparticles.

When you spoke about size in the range of up-to 2 µm flashing with DAPI staining, this advocates for the presence of apoptotic bodies, that retain some DNA and would be stained with DAPI. You may want to try other cell-permeant DNA stains like SYTO xx or DRAQ5 (red fluorescence under 488 nm excitation) to check, or even cell-impermeant stains (e.g. Propidium iodide) since your material has been fixed with PFA.

Other option which comes to mind would be Weibel-Palade bodies, which then are vey specific for endothelial cells (not present in other cell types) ... These would stain using anti-VWF antibodies. For micrograph example of WPB, see George F et al, Demonstration of Rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean spotted fever. Blood. 1993 Oct 1;82(7):2109-16.

Hope that helps. Philippe

Thanks for the thread.  We have thought of apoptosis and Weibel Palade bodies, but neither makes sense. The DAPI positivity is rare in these bodies and the cells do not show any signs of apoptosis (i.e. they were attached, well structured, mostly confluent HUVECs). The other issue we are having is that the particles are autofluorescent, which doesn't make sense for either apoptosis or weibel palade bodies (ie usually these events do not cause fluorescent bodies to form).  The autofluorescence seems to peak in the yellow range, not the green, further complicating this observation, as typically autofluorescence because of cellular break down peaks in the green. Any other ideas would be appreciated.  

Hi Daniel

Im using also HUVECs from Lonza and Im getting unspecific signal around the nucleus when I only incubate with the secondary antibody. I try different secondary antibodies wavelenght ( red, green), different companies (Alexa Fluor, Jackson) , different blockings (2%BSA, 5% normal goat serum), different fixations (methanol, 2%PFA) and the unspecific signal only decreased when I blocked with 5% normal goat serum. I will perform an immunofluorescence experiment without no primary and secondary antibodies and see if I get the unspecific signals around the nucleus wich means that HUVEC might have autofluorescence. I will let you know my results

Thanks. Quick question, is your auto fluorescence dots around the nucleus and if so what size?

I cant precise the size but I attached some pictures

So without anything HUVEC shows autofluorescence in the green.

The other cell line I work is HMEC (Human Microvasculature endothelial cells) and they show also autofluorescence.

I also changed some conditions for my negative controls and the signal was reduced.

Fixation: 2%PFA, 15min, room temperature

Incubation with 0.3M Glycine in PBS1x, 10min, room temperature, shaking

Blocking with 10% normal serum goat in PBS 1x, 1h, room temperature, shaking ( I bloked in serum goat because my secondary antibody is raised in goat)

Incubate 2nd antibody goat anti-rabbit , 30min, room temperature, shaking

Mount in medium with DAPI

Thanks, and yes those appear to be the same autofluorescent dots I get in my ATCC HUVECs. I have not been able to remove them completely even when I only fixed in 1% PFA and them directly mounted with DAPI, so they don't seem to be related to the antibodies.  I am very interested in these as I see them in my patient samples as well, but have not been able to identify them yet. Any ideas? 

These are fluorescent lipofuscins or aggresomes (Google both). There are often related to cell senesecence and quite common in long lived cells such as myocytes.  If they increase with protease inhibitors they are more likley aggresomes.  If you can reduce them with a little borohydride they may be lipofuscin.

They should be more in aged patients samples.

Good luck.

Thank you for the advice. I have thought of both, but I don't know the best way to positively identify them, as both don't seem to have good markers. I have tried a number of experiments based on previous recommendations (i.e. Weibel-palade markers, apoptotic markers, neuropilin) but have not gotten positive results. 

For my samples I did not use any protease inhibitors. I have tried to reduce them with borohydride but they don't quench. The other strange thing is that the cancer patient samples they seem to have different peak fluorescences (i.e. in some patients they peak in green and in others they peak in yellow/orange) Do you have any advice on proper identification for either lipofuscin or aggresomes?  

You are in new territory.  Try to interfere with protein recycling.  Inhibit proteases, proteosomes and see if perinuclear  autofluorescent specks grow.

You could also check if  telomerase length correlates to fluorescence in a cultured population.

Only MS can determine what these autofluorescent specks really are. If you can figure out a way to concentrate these from cell lysates, my group  or many others could help you identify composition.

Dr. Latif: I suspect you dont see autofluorescent specks because you are using fresh isolates. Do you have any HUVECs passaged 5-7 times? A percentage of them may show perinuclear autofluorescent bodies (always near microtubule-organising center (MTOC).

These specks are hotly fluorescent (high quantum efficiency comparable to GFP or phycoerythrins) and do not necessarily require fixing. Pretty much unaffected by methanol/acetone. I have seen them unstained, in live cells (HVSMC, A549, SAEC) and commonly in cryo sectioned  tissue (eg. adult heart).

Do you see these specks around the cells as well? I seem to have similar spots on HMEC-1 cells and i also find them surrounding the cells at times?

PFA gives autoflourescence in green filter....

Please check that. U can reduce the autoflourescence of PFA by including glycine in your washing buffer. Please check the Conc. in appropriate papers.

I have also seen these artefacts in living and fixed Huvec cells.