I need to stain immune cells such as T, B and DC in mice, and to do so, I am conducting titration. But I wonder if I need to do titration for each organ. In other words, can I use the concentration from titration in spleen for lymph node and blood?
I am asking because some cells like DC and NK don't exist enough for titration in LN. I haven't tried, but I may face the same problem for blood samples too.
For spleen obviously you ll need. And for LN and blood I don't think you need. But why don't you go by simple method at first and follow the desired protocol of basic immunology for staining T, B and DC cells.You can consult the basic immunology protocol book as such to clear your doubts.
Hi Eri.
Ultimately, it's best to create one cocktail of antibodies that's compatible for all the sources you wish to use. Titrations can be tricky, but unless you're conjugating your own antibodies, most companies offer a 'recommended' concentration to use per 1 million cells. This assumes that you have a homogeneous population, which is almost never the case.
Some could certainly argue the case for a sepcific titer to each organ. However, my logic is based on my own experience that, the less variables I have, the easier it is to figure things out when something goes wrong. Especially when screening multiple sources at one time, you now have the consistency of one titration.
I have found that, if I titer my antibodies against splenocytes, they generally work as well with blood, liver, lymph node, and even thymus. The numbers of your cells of interest will certainly be proportionaly lower or higher, depending on the source, but, so long as your wash steps are thorough and the samples are generally treated the same, the only variablitly you should see is the MFI of some of your markers due to different levels of expression (assuming you're going beyond staining for just lineage).
After all that, I too agree that following the simplest methods are the way to go...at frist.
Best of luck.
Hi,
What I could gather from your question is that you need to titrate 3 antibodies. Say for example, CD3 FITC (T cells) CD19 PE (B cells), CD11c APC.(DCs). What I would normally do is, I will use mouse spleen cells for titrating CD3 and CD19 antibodies and LN cells for titrating CD11c antibodies. You will find more DCs in LN, so use cells from LNs while titrating for CD11c. Having said that, there is no rule that you should only use LN cells to titrate CD11c, As I prefer to see good MFI peaks in my dilutions I chose an organ where I could find more cells which express my marker.
Thank you all for answering my question. After all, I used splenocytes to titrate and used the concentration for LN. When I tried with CD8, the result of titration from splenocytes, LN, and blood were all the same. So, using another tissue seems to be working.
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