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Jahnmatz

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  1. Hi Eunice I will gladly help you with you. However, I think that I need some more information about you assay first. As I understand it you are seeing antigen-specific IgG and IgM spots in ELISpot, but not in FluoroSpot when you run a dual IgG/IgM assay? Is that right? Since you see IgG and IgM spots in the total wells, there seems to be no problem with the detection antibodies or the cells. Please provide some more info on the protocols (you dont have to share specifics, only the general protocol that you use). Best regards Peter
  2. IL-2 specific activity

    Hi! The IL-2 supplied in the ELISpot kit is used for stimulation of memory B cells in combination with R848. The activity of the IL-2 is confirmed to give more than 500 spots per 50 000 PBMC (per well) with pre-stimulation using 10 ng of IL-2/ml and 1 ug/ml of R848. The biological activity of IL-2 has not been compared with an international reference reagent to establish the number of units. Best regards! Peter
  3. ELISPOT B cell IgG background and no spot

    Hi again Did you use any negative control for IgG? If so what? It seems that there is something in your wells that gets captured by the coating antibody and is then captured by the detection antibody eventually leading to this background in IgG. Often, this "something" comes from the cell suspension, either in the media or some wash-step that contained IgG. It should not be the anti-CD40-mAb since this is most likely a mouse antibody, right? And I guess that before you add your cells to the plate, you had washed them a couple of times right? On your plate, is there a control well without cells but only coating antibody and detection antibody? Best regards Peter
  4. ELISPOT B cell IgG background and no spot

    Hi! Im sorry that you got this problem 3 times in a row. I will help you as much as possible and im sure that we can sort this out! First, I have some questions: 1. What type of cells did you use? How were they stimulated? 2. What type of controls did you have, how was the results there? 3. What is the difference between well G7 and G8. There seems to be no background in G7, but a lot in G8. Best regards Peter
  5. Antigen-specific B cell ELISPOT

    Hi Akira. I could probably help you with this. 1. The detection antibody for IgG (MT78/145) and IgM (MT22) could be used at 0.5ug/ml (1:1000 dilution). 2. If you noticed that the frequency of antigen-specific B-cells were low, I would recommend using non-sorted PBMC (250-500k cells/well). Since the recovery of purification is not 100%, there is a possibility that you loose important cells in the process. If you want to look at resting MBC, I would recommend pre-stimulation of PBMC for 5 days using R848 and rec.IL-2 before use in the ELISpot. I have attached an article describing this protocol for stimulation (although for FluoroSpot). Dont hesitate to ask if there is something else. Good luck! Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.pdf
  6. Hi Tyler. Franco already beat me to answering your question. I think its a great idea to test the supernatant of your stimulated cells in ELISA to see if you have ag-specific ab there to begin with. ELISA should be sensitive enough to detect low concentrations of these ag-specific abs. As a complement to Francos answer about the stimulation protocol, I would recommend using our protocol with 1µg/ml R848 and 10ng/ml rec.IL-2, BUT instead of stimulating them for 3 days, you stimulate them for 5 days. This will give rise to a greater clonal expansion of your B cells compared to what you get after 5 days. I use this protocol when looking at Ag-specific MBC that exist in extremely low frequencies in circulation. I have attached the results of the small comparison of B-cell stimulation after 3, 5 and 7 days with R848 and IL-2. Please let me know if you need these stimulis, I know that there is a guy close to you that works with this protocol. Best regard Stimulation time.pptx
  7. Dear Mazor I have looked into the your very interesting question and found a possible way to identify both memory B cells and plasma cells from your samples. Like Christian said, Plasma cells are usually found in the bone marrow after the infection has been cleared, but they can also be found in the spleen of mice during an infection. Recently, antibody-producing plasma cells have also linked as regulators of the immune response. In this review, they have focused on the cytokines produced by plasma cells in mice: http://www.sciencedirect.com.proxy.kib.ki.se/science/article/pii/S0952791514000375. Pretty cool stuff, but it is hard to distinguish between long-lived plasma cells and memory-B-cell plasma cells in this context. I have not yet identified a cytokine that is only secreted by activated memory B cells that has differentiated into plasma cells but not long-lived plasma cells, or the other way around. However, since your problem is an interesting one, I will continue looking.
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