Christopher,

you might want to contact my colleague, Christian Hennig, at http://zellkraftwerk.com/

They are doing chipcytometry. I could imagine screening of your cells with their library of anti-human antibodies. I have one publication using chipcytometry (about human basophils in Nat. med. :-) ) with Dr. Hennig.

I do not know about the costs of the service, but I think we agree that characterizing your cells is an important thing to do before doing the biggest experiments with the monkeys...

Strange actually, that those classical (human) plasma cell markers do not work for your monkey cells.... I use exactly these markers on CpG-incubated B cells (5d culture). Works perfect, although I think these markers are not completely accurate (to distinguish between activated memory cells, plasma blasts and plasma cells). I use the CD138 from Miltenyi. The BD antibody does not work for me.

Bye from Hannover

Hi Dorothea,

Thanks for the good comments! So the CD138 from miltenyi works for rhesus macaques, you say? I've also only tried the BD one, which does not work for me.

When using CD38 and CD27 I also cannot distinguish differentiated plasma cells (via stimulations). Which stain do you use for this?

Best,

Christopher

Dorothea's suggestion sounds interesting.

Alternatively, you could try using all the antibodies found in a lineage cocktail (http://www.bdbiosciences.com/external_files/pm/doc/tds/stem_cell/live/web_enabled/562722.pdf) except the ones known to bind b-cells / plasma cells. They have at least 2 different lineage cocktails for humans. I would presume that they cross react with rhesus epitopes, but I have not done a test to find out.

Evan, thanks for the link. About 85% of the Abs in the cocktails cross-react, although some additional markers would need to be added as specificities are not always 100%. A good lead though, thanks!

High Christofer,

X-reactivity of MAbs with animal cells is not always straightforward even for primates (somehow closer to humans). Except for chimpanzees, lack of x-reactivity is rather frequent. In my (short-term) experience looking for X-reactivity of anti-human platelet markers with monkeys, cats, mini-pigs, dogs ..., I observed very heterogeneous behavior for MAbs being part of the same human CD. Trying one single (e.g. CD38) clone is certainly not the way to go in your particular case.

Thus, I would suggest you i) to extend your question on several forums (this and others, including vet-oriented) to get more responses, ii) not hesitate to screen several MAbs per CD (from diverse vendors - you may ask for special commercial offers if you are to buy small amounts of many MAbs; trick: you may also be able to grasp samples of near-to-peremption date commercial reagents for lower price), iii) use indirect IF rather than conjugated MAbs (much cheaper and you can devise staining experiments on 96 microplate formats to enlarge your screen) iv) look for vet-oriented litterature (e.g. TNO in Rotterdam, NL is a huge primate experimental cter) , v) have a deeper look in HLDA (Human Leukocyte Differentiation Antigens) books = those international workshops where worldwide scientists screened next-to-become CD MAbs. They include over 8 volumes and I remember you can get whole chapters on non-human primates studies. Just an idea: you may visit website of AbDbiotech where they have a bunch of CD MAbs checked for X-reactivity with animals, including (I think) rhesus.

Best, PP

Hi Christopher, sorry I forgot to mention that I work with human cells!

CD19-PECy7, CD27-APCCy7, CD38-PerCPCy5.5, CD138-PE. I incubate isolated B cells for 5 days (with and without other cells, therefore I need this complete cocktail) with 0.1 µM CpG. On a Canto II I first gate on CD19+ cells, then on the CD27hi, from these, a proportion is CD38 and again a smaller proportion are CD138+ (plasma cells). I kind of keep to the definition for plasma cells from the literature... One could also gate on the CD38/CD27 expressing cells and check how many of them have CD138. If things do not work at all you can send us frozen cells and we could do the staining in parallel with our own (human) cells. By the way, we also order antibodies from Biolegend, they mostly work well and are less expensive.

Hi Dorothea,

Thank you very much for the offer. However, our main problem is not really the staining procedures. We can stain human cells just fine. It's the non-reactivity of certain clones in non-human primates that is the problem (combined with what seems to be slightly different expression patterns). E.g. the CD19 Ab (clone HIB19 sold by Biolegend and most other companies is non-reactive). I appreciate the help though!

HI Christopher,

You are correct CD19 is generally not recognized in NHP with human mAb.

Here is a link to a database of mAb and their reactivity to several non-human primate species.

http://nhpreagents.bidmc.harvard.edu/NHP/ReagentList.aspx

You will see that for the same antigen, different clones exhibit different recognition pattern related to polymorphism between species. Keep in mind also that expression pattern may differ between human and the primate species of interest and initial testing (clone, concentration) is always warranted before major experiments.

Good luck!

Hi Bernard,

Thanks for the link and suggestions! I use that site very frequently and always check for possible clones there first if I plan some new stainings. I agree it's not only the problem of clones being reactive or not. There is also the problem of different expression of surface markers between NHPs and humans. That's the main reason for the difficulty of setting up a good plasma cell staining protocol.

I've finally decided to go for using a very extensive dump and a few positive markers though. Hope it will work!

Hi Christopher,

We are troubleshooting something similar at the moment. We have used both the BD CD138-APC (MI-15) and ebiosciences clone (DL-101) to stain plasma cells in the bone marrow of NHP, and get pretty good separation. The CD138+ plasma cells population we find is IgG-, IgD-, IgM-, CD20- and CD19lo/-. We generally find that both CD27 and CD38 staining on NHP B cells is a little funny and not comparable to what we see in human samples. Beckman coulter make a CD19 antibody Clone J3.119 that usually works in NHP, although the separation is less than observed in human samples.

Are your staining fresh or frozen BM samples? Many people have suggested that freezing the BM samples can significantly reduce/eliminate CD138 staining.

Good luck.

Hi Adam,

I'm glad to hear the CD138 works for you. I've tried the BD one before but didn't see anything then. I did try on frozen cells so maybe that could be the reason why the stain was negative. CD19 works so/so for us. It seems we have slightly worse separation than what I've seen in other NHP papers, and I believe it could be because we work mostly on Chinese rhesus and not Indian as is most common.

Did you try to stain the CD138+ for intracellular Ig as well? or sort into ELISpot wells?

When you say that CD38 and CD27 are funny. Do you mean that they are not high as in humans or that they have a quite spread expression (in the CD138+ gate)?

Thanks for the help!

We now have a paper on this where Paola has characterized a number of markers that can be used in conjunction with CD138 to identify PCs.

https://www.ncbi.nlm.nih.gov/pubmed/27446073

CD19 is enough 

As I understand it, there are both CD19+ and - BM PCs, so not so sure that will be enough. At least in humans. Also, the CD19 marker from macaques is pretty poor. Especially for rhesus macaques of Chinese origin ones, it seems.

sorry I didn't see the original question you made in 2013. I have no experience with macaques, however CD19 would theoretically work fine for all species.

what do you mean by PCs in BM exactly? why would you measure PCs in BM? in other words, how do you generate PCs? 

No worries. This thread mainly talks about macaques. BM refers to bone marrow. The PCs are not in vitro generated or anything, they are from macaque bone marrow aspirates.

This is what i thought just wanted to confirm. In vivo is different because in vitro I would rather talk in terms of CD19 high/low no negatives. So what I said above won't apply here you would certainly need more than one marker. I was actually looking for markers that would work after in vitro stimulation and I thought of CD138 too but not very sure I will just look up the thread again to see what you propose. Thanks Chris

Ah. That makes it more clear. Yes, CD19 might be enough then. I don't have very much experience of in vitro derived PCs. Based on some old experiments we did, we used CD27++ CD38++ to denote PCs. It's a very clear distinction with those two markers.

Agree with you. Look this review

                        https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467643/

                        plasma cells      IgDneg  CD27high CD38high CD138+

                        plasmablasts      IgDneg   CD27high CD38high CD138neg

Something else the idea about "in vitro activation" is that you evaluate the activation from day zero so you can use CD19 as sole marker for "purified B cells" not PBMCs or Bone marrow. On the other hand the CD19 neg B cells (or PCs) should be a very very small fraction that is why CD19 can be still useful as sole marker in your experiment. So my question to you what was the % of PCs CD19 neg in your experiment?

CD19 is such a crap marker in Chinese rhesus macaques (or more accurate. The Ab that is currently found to be semi-cross reactive), so the frequency of +/- is not that clear, especially in complex samples, such as bone marrow or PBMCs.

Thanks for the information. I will keep this point in mind and update you if I found something else.