Dendritic cell immunology - activated naive cells? (May/22/2009 )
I'm new to DC immunology so please help!
I isolate bone marrow dendritic cells from mice femurs, culture (RPMI + GM-CSF (5-10 ng/ml) in petri dishes or T25) and than eventually stimulate them with LPS. The problem is that my naive cells seem to be already or just as activated as the cells that I've stimulated. I've tried to culture them on various days ie. d6, d8 and d10 and still get the same result. Everything should be pyogen free. I thought isolating bone marrow dendritic cells would ensure that I have naive cells. Are they attaching to something? Has anyone tried culturing them on teflon plates?
Also, I was having trouble detecting CD40 expression on the activated DCs by Flow cytometry. CD40 should be expressed on the cells but is there a better way to detect CD40 upregulation in DCs?
DC Girl on May 22 2009, 05:01 PM said:
Are you sure the problem is that the "naive" cells are already activated, or that the supposedly "active" cells are in fact still naive? What measure are you using to detect activation? CD40 expression?
As to detecting CD40, there are certainly many other ways to detect it... ELISA would be the first that comes to mind.
-Carlton
Carlton H on May 24 2009, 02:21 PM said:
DC Girl on May 22 2009, 05:01 PM said:
Are you sure the problem is that the "naive" cells are already activated, or that the supposedly "active" cells are in fact still naive? What measure are you using to detect activation? CD40 expression?
As to detecting CD40, there are certainly many other ways to detect it... ELISA would be the first that comes to mind.
-Carlton
To detect activation, I look for the expression of CD80/CD86 by Flow after O/N stimulation with LPS. The unstimulated cells show upregulation of CD80/86 also. These naive cells were isolated on d6 so I was assuming that they would still be fairly immature and naive. Would the GM-CSF activate the DCs? I noticed that some protocols add IL-4 to the media but I don't. I wasn't sure if I needed to since I wanted immature DCs.
As for CD40, I was hoping that it would suggest Th1 response by Flow but I was unable to detect any. I will use ELISA to look at IL-12 and IFN-gamma.
DC Girl on May 25 2009, 09:30 PM said:
Carlton H on May 24 2009, 02:21 PM said:
DC Girl on May 22 2009, 05:01 PM said:
Are you sure the problem is that the "naive" cells are already activated, or that the supposedly "active" cells are in fact still naive? What measure are you using to detect activation? CD40 expression?
As to detecting CD40, there are certainly many other ways to detect it... ELISA would be the first that comes to mind.
-Carlton
To detect activation, I look for the expression of CD80/CD86 by Flow after O/N stimulation with LPS. The unstimulated cells show upregulation of CD80/86 also. These naive cells were isolated on d6 so I was assuming that they would still be fairly immature and naive. Would the GM-CSF activate the DCs? I noticed that some protocols add IL-4 to the media but I don't. I wasn't sure if I needed to since I wanted immature DCs.
As for CD40, I was hoping that it would suggest Th1 response by Flow but I was unable to detect any. I will use ELISA to look at IL-12 and IFN-gamma.
Hi DC girl, if you could send a detailed protocol of what you do I might be able to give you some help. Also, any chance we can have a look at your FACS plots to see what you mean by up-regulation in the naive samples.
cheers
thanks for interest. here's the protocol...
D0 = the dendritic cells are isolated from mice femurs and cultured in RPMI + GM-CSF (5-10 ng/ml) in petri dishes or T25
D3 = add 10 mL media if cultured in petri dish (T25 plates = remove media, discard and add fresh media)
D6 = change media for D8 (remove 10 mL and fresh 10 mL media) or collect and count cells to check for CD11c-PE by Flow and for stimulation.
Stimulation:
0.4x10^6 cells (in media) incubated with LPS (10 ng/mL) O/N @37C in 24 well plates
Flow:
collect and count cells (use 0.3x10^6 cells/antibody)
block 30 min (on ice in the dark)
add CD86-PE 30 min (on ice in the dark)
check on FACS cantoII
I'm also new to the FLOW software, we use FloJo which I am just starting to get the hang of. Any tips?
Hmm... You got me. I'll keep thinking about it but all I have to add at this point is that GM-CSF should not be stimulating the dendritic cells.
DC Girl on May 27 2009, 09:26 PM said:
D0 = the dendritic cells are isolated from mice femurs and cultured in RPMI + GM-CSF (5-10 ng/ml) in petri dishes or T25
D3 = add 10 mL media if cultured in petri dish (T25 plates = remove media, discard and add fresh media)
D6 = change media for D8 (remove 10 mL and fresh 10 mL media) or collect and count cells to check for CD11c-PE by Flow and for stimulation.
Stimulation:
0.4x10^6 cells (in media) incubated with LPS (10 ng/mL) O/N @37C in 24 well plates
Flow:
collect and count cells (use 0.3x10^6 cells/antibody)
block 30 min (on ice in the dark)
add CD86-PE 30 min (on ice in the dark)
check on FACS cantoII
I'm also new to the FLOW software, we use FloJo which I am just starting to get the hang of. Any tips?
Hi DC girl.
Do you only look at CD86? what about CD80, CD40 and MHC II. what about cytokines?
I agree your cells dont look too stimulated, but there's definitely difference with the unstimulated ones. I'll need to see CD40 and MHC-II to really agree with your statement that the unstimulated cells look activated. CD86 is not a discreat peak and although there's not a big shift after your LPS treatment, there's definitely a change in the population, and more cells seem to be expressing higher CD86 (this is not an all or nothing, unstimulated cells have CD86 in the membrane).
With CD40 and CD80 the difference is usually a much clearer -ve to +ve (no peak, to bright peak).
Class II on the other hand will show a "spread" histogram (indicating different levels of expression in different cells) that shifts strongly to the right in a sharp peak after stimulation.
I suggest you reanalyse the plots as I've indicated with the red lines as that's the popluation that's changing. Maybe even look at a dot plot to see proper populations.
Also, you should probably do a titration of your LPS, 10ng/ml might be a bit too low, we used to use 100ng/ml.
Finally, how do you recover you cells from the petri dishes, or T25 before you plate them in 24 well plates? You have to be quite gentle with them for the cells not to get activated.
Hope this helps, any more questions ask
almost a doctor on May 28 2009, 05:00 AM said:
DC Girl on May 27 2009, 09:26 PM said:
D0 = the dendritic cells are isolated from mice femurs and cultured in RPMI + GM-CSF (5-10 ng/ml) in petri dishes or T25
D3 = add 10 mL media if cultured in petri dish (T25 plates = remove media, discard and add fresh media)
D6 = change media for D8 (remove 10 mL and fresh 10 mL media) or collect and count cells to check for CD11c-PE by Flow and for stimulation.
Stimulation:
0.4x10^6 cells (in media) incubated with LPS (10 ng/mL) O/N @37C in 24 well plates
Flow:
collect and count cells (use 0.3x10^6 cells/antibody)
block 30 min (on ice in the dark)
add CD86-PE 30 min (on ice in the dark)
check on FACS cantoII
I'm also new to the FLOW software, we use FloJo which I am just starting to get the hang of. Any tips?
Hi DC girl.
Do you only look at CD86? what about CD80, CD40 and MHC II. what about cytokines?
I agree your cells dont look too stimulated, but there's definitely difference with the unstimulated ones. I'll need to see CD40 and MHC-II to really agree with your statement that the unstimulated cells look activated. CD86 is not a discreat peak and although there's not a big shift after your LPS treatment, there's definitely a change in the population, and more cells seem to be expressing higher CD86 (this is not an all or nothing, unstimulated cells have CD86 in the membrane).
With CD40 and CD80 the difference is usually a much clearer -ve to +ve (no peak, to bright peak).
Class II on the other hand will show a "spread" histogram (indicating different levels of expression in different cells) that shifts strongly to the right in a sharp peak after stimulation.
I suggest you reanalyse the plots as I've indicated with the red lines as that's the popluation that's changing. Maybe even look at a dot plot to see proper populations.
Also, you should probably do a titration of your LPS, 10ng/ml might be a bit too low, we used to use 100ng/ml.
Finally, how do you recover you cells from the petri dishes, or T25 before you plate them in 24 well plates? You have to be quite gentle with them for the cells not to get activated.
Hope this helps, any more questions ask
Hey AAD!!
I really appreciate the help and disscussions so far.
1)I do look at CD80 also but I find that it is more difficult to see a difference (I am assuming now that unstimulated cells also have CD80 on their membranes). The CD86 data usually looks better. I've only tried CD40 once and it did not show anything!! No change from unstained control. I didn't do an antibody titration yet but does this antibody require a larger amount to see expression by Flow? MHC-II is on back order. LOL...and I'm working on ELISA for IL-12 and IL-4. I was trying to set the conditions separately for each antibody before I tried the four all at once. Eventually I'd like to look at 6 but I've never used FLow before in my life...so I thought I'd start small. The lab I work in are mainly biochemists and chemists so they can't help me much.
2)recovery of cells from:
(A) petri dishes - collect cells by pipetting into a falcon tube before centrifugation
(
T25 - gently pipetting up and down 4X before centrifugation (this was to remove lightly adhered cells and was supposed to be a method that would ensure purity of DCs over quantity like in method A)I think that I may have over handled them.
Have you ever tried serum-free media for BMDCs? I was thinking of looking at some macrophage cell lines also (like IC-21 or J774A) but I've heard that those are usually activated. Do you think it's worth my time? Should I stick to primary cell culture- it just seems so slow.
Thanx
DC Girl on May 29 2009, 11:02 AM said:
almost a doctor on May 28 2009, 05:00 AM said:
DC Girl on May 27 2009, 09:26 PM said:
D0 = the dendritic cells are isolated from mice femurs and cultured in RPMI + GM-CSF (5-10 ng/ml) in petri dishes or T25
D3 = add 10 mL media if cultured in petri dish (T25 plates = remove media, discard and add fresh media)
D6 = change media for D8 (remove 10 mL and fresh 10 mL media) or collect and count cells to check for CD11c-PE by Flow and for stimulation.
Stimulation:
0.4x10^6 cells (in media) incubated with LPS (10 ng/mL) O/N @37C in 24 well plates
Flow:
collect and count cells (use 0.3x10^6 cells/antibody)
block 30 min (on ice in the dark)
add CD86-PE 30 min (on ice in the dark)
check on FACS cantoII
I'm also new to the FLOW software, we use FloJo which I am just starting to get the hang of. Any tips?
Hi DC girl.
Do you only look at CD86? what about CD80, CD40 and MHC II. what about cytokines?
I agree your cells dont look too stimulated, but there's definitely difference with the unstimulated ones. I'll need to see CD40 and MHC-II to really agree with your statement that the unstimulated cells look activated. CD86 is not a discreat peak and although there's not a big shift after your LPS treatment, there's definitely a change in the population, and more cells seem to be expressing higher CD86 (this is not an all or nothing, unstimulated cells have CD86 in the membrane).
With CD40 and CD80 the difference is usually a much clearer -ve to +ve (no peak, to bright peak).
Class II on the other hand will show a "spread" histogram (indicating different levels of expression in different cells) that shifts strongly to the right in a sharp peak after stimulation.
I suggest you reanalyse the plots as I've indicated with the red lines as that's the popluation that's changing. Maybe even look at a dot plot to see proper populations.
Also, you should probably do a titration of your LPS, 10ng/ml might be a bit too low, we used to use 100ng/ml.
Finally, how do you recover you cells from the petri dishes, or T25 before you plate them in 24 well plates? You have to be quite gentle with them for the cells not to get activated.
Hope this helps, any more questions ask
Hey AAD!!
I really appreciate the help and disscussions so far.
1)I do look at CD80 also but I find that it is more difficult to see a difference (I am assuming now that unstimulated cells also have CD80 on their membranes). The CD86 data usually looks better. I've only tried CD40 once and it did not show anything!! No change from unstained control. I didn't do an antibody titration yet but does this antibody require a larger amount to see expression by Flow? MHC-II is on back order. LOL...and I'm working on ELISA for IL-12 and IL-4. I was trying to set the conditions separately for each antibody before I tried the four all at once. Eventually I'd like to look at 6 but I've never used FLow before in my life...so I thought I'd start small. The lab I work in are mainly biochemists and chemists so they can't help me much.
2)recovery of cells from:
(A) petri dishes - collect cells by pipetting into a falcon tube before centrifugation
(
T25 - gently pipetting up and down 4X before centrifugation (this was to remove lightly adhered cells and was supposed to be a method that would ensure purity of DCs over quantity like in method A)I think that I may have over handled them.
Have you ever tried serum-free media for BMDCs? I was thinking of looking at some macrophage cell lines also (like IC-21 or J774A) but I've heard that those are usually activated. Do you think it's worth my time? Should I stick to primary cell culture- it just seems so slow.
Thanx
Im having the same problem of DC girl and im starting to think that all the literature about DC activation are wrong
I have done everything but always get the same results.
Bone marrow DCs always look a little bit matured (compared to DCs freshly isolated from the spleen).
Just the act of replating DCs into fresh plastic will induce some levels of maturation due to the integrin signaling involved with interacting with the plastic. This can result in cytokine production and UCM (upregulation of costimulatory molecules. When making BMDCs, I have found it to be very helpful to have a separate plate that I only use for monitoring DC development. I have found that just moving the cultures causes some levels of maturation and UCM as the cells move and then engage with the plastics.
I have also found that certain markers can be "difficult", getting CD40 for a figure was a major pain in the neck. I had the best results if I used DCs when they were around 70% CD11c +. If they went too long in culture, they started to get less responsive to stimulation.
Hope this helps!