Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Dendritic Cells and Flow Cytometry


  • Please log in to reply
11 replies to this topic

#1 immunologist

immunologist

    member

  • Members
  • Pip
  • 6 posts
0
Neutral

Posted 07 April 2009 - 08:38 PM

I am working with dendritic cells and I am trying to determine the expression of cell surface markers IA [mouse MHC II],CD80 and CD86 using flow cytometry.
I bought antibodies for the above mentioned markers from ebiosciences and tried it on splenocytes to check the antibodies. It worked perfectly fine. When I tried using my dendritic cells [ bone marrow derived and JAWSII cell line], initilally it did stain for IA,CD80 and CD86 but it was much lower than what was expected in Dcs stimulated with LPS.
I tried different concentrations of LPS to stimulate Dcs but it did not work. From the recent data that I have obtained, I am not getting any/ significantly low populations of IA positive /Cd80/CD86 DCs that have been stimulated with LPS. I even tried using protease inhibitors to check whether that had any effect, but it did not. Initially I used to fix the cells after staining using 10% buffered formalin but I realized that it might be too concentrated so I used 1% buffered formalin. That also did not help.
I am really frustrated because I am unable to proceed with any of my work due to this.
If anyone has any clue or idea about this , I would greatly appreciate if you could help.


Thank You.

#2 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 181 posts
1
Neutral

Posted 08 April 2009 - 02:55 AM

Hi there :)

How long are you culturing your BM cells after isolation and in what kind of media/cytokines are you using?

Clare

#3 immunologist

immunologist

    member

  • Members
  • Pip
  • 6 posts
0
Neutral

Posted 08 April 2009 - 07:52 AM

Clare,
I culture them for ten days in RPMI media [complete] with 10ng/ml GM-CSF.

#4 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 181 posts
1
Neutral

Posted 09 April 2009 - 02:11 AM

Clare,
I culture them for ten days in RPMI media [complete] with 10ng/ml GM-CSF.



I'm no DC expert but when I have cultured DCs from BM I would culture them in media (KDS RPMI +2-ME +10% FCS) with Flt3-L (also for 10 days). And the FACS staining was always nice and DC-like (i.e: I used markers like CD45RA,CD11c, CD11b and HSA).

#5 immunologist

immunologist

    member

  • Members
  • Pip
  • 6 posts
0
Neutral

Posted 09 April 2009 - 09:14 AM

So you do not use GM-CSF at all? Are these DCs in an immature state?

#6 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 181 posts
1
Neutral

Posted 14 April 2009 - 03:06 AM

So you do not use GM-CSF at all? Are these DCs in an immature state?


Nope. Just Flt3-L as suggested by the DC lab (Ken Shortman's) at my old institute. I would get pDC as well as conventionals (both CD8+ and CD8-). That's all I looked at though.

Clare

#7 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 14 April 2009 - 04:03 AM

So you do not use GM-CSF at all? Are these DCs in an immature state?


Nope. Just Flt3-L as suggested by the DC lab (Ken Shortman's) at my old institute. I would get pDC as well as conventionals (both CD8+ and CD8-). That's all I looked at though.

Clare



If I remember correctly from my DC days, BM cells in the presence of Flt3-L will differentiate mostly into plasmacytoid DCs, while BM cells in the presence of GM-CSF will differentiate mostly into myeloid DCs.

I always used GM-CSF for 10 days, and got ~90% CD11c+ cells.
Can you tell exactly what protocol you use to get DCs from BM (plates, volumes, feeding), and also how do you stimulate the cells (when, how long, again volumes, plates), and how do you stain them.

Cheers.

#8 Clare

Clare

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 181 posts
1
Neutral

Posted 14 April 2009 - 04:15 AM

YAY! I knew a DC person would come along eventually ;)

Clare

If I remember correctly from my DC days, BM cells in the presence of Flt3-L will differentiate mostly into plasmacytoid DCs, while BM cells in the presence of GM-CSF will differentiate mostly into myeloid DCs.

I always used GM-CSF for 10 days, and got ~90% CD11c+ cells.
Can you tell exactly what protocol you use to get DCs from BM (plates, volumes, feeding), and also how do you stimulate the cells (when, how long, again volumes, plates), and how do you stain them.

Cheers.
[/quote]

#9 immunologist

immunologist

    member

  • Members
  • Pip
  • 6 posts
0
Neutral

Posted 27 April 2009 - 08:31 AM

Thanks for replying.

I plate bonemarrow cells at a density of 10^6 cells/ml in 10 ml RPMI1640 with 10ng/ml GM-CSF [100x15mm plate].
On day three I remove non adherent cells and add fresh medium. Onday seven , I change medium and use the cells on day 10.
I treat them with 5 microgram/ml LPS for 24 hrs to stimulate them.
I do not see any morphological changes, they do not produce il12. They don't even express Ia,CD80 or CD86.


For staining, I used to suspend them in PBS-BGS-Na azide [1ml, 10^6 cells]. Add 30microliter or 12microlit of PE or FITC conj abs.
Wash, fix with 10% buffered formalin [now I use 2.5%].

#10 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 28 April 2009 - 06:35 AM

Thanks for replying.

I plate bonemarrow cells at a density of 10^6 cells/ml in 10 ml RPMI1640 with 10ng/ml GM-CSF [100x15mm plate].
On day three I remove non adherent cells and add fresh medium. Onday seven , I change medium and use the cells on day 10.
I treat them with 5 microgram/ml LPS for 24 hrs to stimulate them.
I do not see any morphological changes, they do not produce il12. They don't even express Ia,CD80 or CD86.


For staining, I used to suspend them in PBS-BGS-Na azide [1ml, 10^6 cells]. Add 30microliter or 12microlit of PE or FITC conj abs.
Wash, fix with 10% buffered formalin [now I use 2.5%].



Hi, I think you might be overstimulating your cells. 5ug/ml sounds like a lot of LPS. I suggest you do a titration of LPS as well as maybe a time course. With 100ng/ml to 1ug/ml of LPS you should get IL-12, IL-6, IL-10.... and upregulation of activation markers, however the levels of this cytokines vary with time, you can detect IL-6 as early as 1h after treatment, it goes up during the first 6h, then goes down and then up again. And I think the same happens for activation markers. You might be taking your cells in the down-regulation phase. You might even be seeing LPS tolerance, which is when your cells became non-responsive to a 2nd LPS stimulation after LPS stimulation. Because there's so much LPS on your treatment, the cells might have time to get activated, and then tolerised in a second stimulation whithin the 24h treatment.
I'll suggest yo try 6h, 18h and 24h stimulation, and also different concentrations, maybe something like 0.1, 0.5, 1 and 5ug/ml. I used to use up to 1ug/ml for some experiments, but most people in my lab used 100ng/ml.

Also, are you stimulating the cells in the 100x15mm plates, or replating them? How many cells are you stimulating? How did you decide to use 5ug/ml for 24h?

Finally, I used to stain my cells (about ~10^6) in as little as 50-100ul. I feel they stain better (although that's probably just my feeling) but you certainly will save loads of antibody.

Hope this helps, any more questions ask.
:D :P

#11 immunologist

immunologist

    member

  • Members
  • Pip
  • 6 posts
0
Neutral

Posted 29 April 2009 - 12:14 PM

I use 24 well plates. I have tried different cell concentrations like 2.5x10^5 to 1x10^6.
I used 5microgram/ml LPS based on some papers that I came across. I even emailed these people regarding the same and
they did not give me any constructive suggestions.

I will def try using low LPS concn and see how my cells behave.Will let u know.


Thanks for the suggestions.

Will bug you when I have more questions. ;)

#12 Spaced_out

Spaced_out

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 15 May 2009 - 11:44 AM

I use 24 well plates. I have tried different cell concentrations like 2.5x10^5 to 1x10^6.
I used 5microgram/ml LPS based on some papers that I came across. I even emailed these people regarding the same and
they did not give me any constructive suggestions.

I will def try using low LPS concn and see how my cells behave.Will let u know.


Thanks for the suggestions.

Will bug you when I have more questions. :)


Your procedure seems to be fine, you are using too much LPS - 10-20 ng/ml is sufficient. But I guess you are lacking the 2nd signal. Either TNF or IFN-gamma would provide the 2nd signal. I hope this helps. The Dc field is real convoluted... :)




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.