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Jens@mabtech.com

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  1. Oh, I see! It looks like the cells have been "pushed" to the side of the well. That might happen if you add the cell suspension first and the stimulus after. In what order are you adding reagents? The best way is to first add medium including (or not including) the stimuli and the cell suspension last. Use for example 50% of the final medium volume for the stimuli (or just medium for negative control) and 50% of the final volume for the cell suspension.
  2. Hej Lena! It looks like you are experiencing the exact same issue as has been discussed in this thread: Briefly, probably some reagent have not reached the bottom of the well. We have seen these blank wells a couple of times, and we have yet to experience a blank well that cannot be "rescued" by repeating the detection steps again. Thus, the problem is not a lack of coated capture antibodies, but a one failed detection step. Please repeat the detection steps in the blank wells as follows: 1. Wash the blank wells 5x with 200ul PBS. 2. Add detection antibody at 1ug/m
  3. Hej Esther! Co-cultures of e.g. cancer cells and effector cells is a quite common setup, although we hardly ever perform these experiments in-house. The important thing is that you find a suitable target:effector ratio and to include control wells with e.g. only the target cells to make sure that they don’t produce the cytokines of interest themselves. For protocol inspiration, you can have a look in this paper by Zuber et al 2005: https://www.ncbi.nlm.nih.gov/pubmed/16005014. Although we do not have extensive experience of using adherent cells in ELISpot/FluoroSpot we have not encou
  4. Hi again Josh, There is actually a paper, Luque et al 2018, in which the authors utilize a one-step detection system for FluoroSpot: https://www.ncbi.nlm.nih.gov/pubmed/30075182 However, they don't detect fluorophore-labeled antigen, but fluorophore-labeled HLA mulitmeres (thus looking for anti-HLA IgG in a transplantation setting). The HLA multimeres might have a higher binding capacity and bind more fluorophore molecules than a single antigen would, potentially increasing the signal, but at least it's some data showing the feasibility of a one-step detection system.
  5. Hi Josh, Welcome to the forum! Sounds like an exciting project! I can't say for sure that it would be impossible, but I think the signal loss might be substantial if you were to utilize direcly fluorophore-conjugated antigens. We have done similar experiments in-house, some of which have been published in Jahnmatz et al 2016: https://www.ncbi.nlm.nih.gov/pubmed/26930550. In this paper, we applied a two-step detection system using peptide-tagged antigens and subsequent anti-tag detection with fluorophore-conjugated mAbs (see attached image). The peptide tags - whose amino ac
  6. Hi E, Great to hear that you have good viability of the cells, and that you are willing try the BCIP/NBT-plus substrate as well as pre-coated plates. Please share your results if you’re up for it! Kind regards, Jens
  7. Hi again E, Thank you for all the details. So, Christian and I have discussed your issue and come up with the following comments: • Perforin is a rather tricky cytokine to detect with good results. If you look in the ELISpot-well photo in this page, you see a visual example of how this system looks after 72h stimulation with a strong CEF peptide responder: https://www.mabtech.com/products/human-perforin-elispot-basic-kit-alp-3465-2a-0. You do get spots after 24h with a good positive control, but for antigen specific responses it is recommended to use 48h or 72h incubation.
  8. Hi E, Would you please provide us with some more information of your protocol to allow us to trouble shoot this problem? I understand that you followed our protocol, but there are details in it that we leave for you as a researcher to decide. For example, what substrate did you use? How long did you develop spots for? How many cells did you add to each well? What was the viability of the cells? What medium did you use? What did you wash the plates with? Kind regards, Jens
  9. Hi T, I think one should avoid using trypsin or accutase as they are both proteolytic enzymes and can lead to the digestion of both the antibodies and the analyte. Instead, we recommend a wash protocol including EDTA. In short, adherent cells are removed by first washing 3x with PBS, and then a 10 minute incubation at 37°C with 100ul PBS supplemented with 1mM EDTA in each well. After that, you just wash once again with PBS, and the membranes should be clean from cells. Please find the steps below and attached as a pdf. Mabtech_ELISpot_adherent_cells_protocol.pdf 1. Wash
  10. Hi M, The maximum sensitivity of ELISpot is hard to say, because in theory it’s unlimited. Let’s say you have an ELISpot well big as a bucket, and you add 100 billion cells in there, and add 1 cell you know are responding to your antigen, then yes, you would theoretically actually be able to detect it. It is a bit dependent on the cytokine secretion over time, as you point out, and that in turn depends on the particular cell and antigen. Unfortunately, we can’t say for sure exactly how much cytokine must be produced to see a spot. But normally, for low-frequent antigen-specific T cell IFN
  11. Hi Flavia, Sorry for the delay in response, you caught us in the middle of our midsummer's holiday. So, before in the experiment with primed+boosted mice you saw false positive spots in the "Ctrl vh +pept" wells, which are now gone. But instead now you have spots in the "Ad-GFP -pept" wells, a type of control you didn't include in your first run, correct? In essence, it looks like the T cells are secreting IFNg without the need for peptide re-stimulation. Could that be a true finding? Yes, it is not surprising. With the prime and the boost you are stimulating the T cells in viv
  12. Great, Flavia! Good luck with your second experiment and please keep us posted! Kind regards, Jens
  13. Hi Flavia and welcome to the Mabtech Forum! I think you are addressing the issue you're having in the best possible way. My guess is that it is the added IL-2 that is causing you false-positive background, so in my view it's good that you are removing it. You shouldn't need extra IL-2 for a short incubation like this anyway. Did you include it in your original protocol for a particular reason? The 1 million splenocytes/well is on the brink of being too many, so you're doing the right thing of decreasing that number to 250k and 500k cells/well. If you didn't have the no-sti
  14. Hi Lily, I hope another Forum-user than me can shed light on your quest for hard evidence papers showing the effect of RBC on ELISpot. As I wrote above, there are theoretical ways of investigating this, but I have yet to see them published in practice. But regardless, for sure, an RBC lysis of whole blood before an ELISpot assay could work out ok, and we have performed it occasionally in-house. But after talking to some of our veteran ELISpot experts here at Mabtech, our official answer is that we would advice against RBC lysis and thus still recommend the Ficoll separation method. B
  15. Hi Sylvain, and welcome to the Mabtech forum! It sounds like a wise choice to go for ELISpot to detect tumor-antigen specific T cells. Here are my answers to your questions: 1. Given that the antigen-specific T cells are very low-frequent, I think you're doing the right thing sorting the T cells first. Normally in ELISpot, you plate between 50k and 500k cells, and in your case I would plate 300k - 400k T cells per well. That way the cells aren't too crowded but you still have plenty of purified T cells to evaluate in each well. 2. The phenomenon of T-to-T cell presentation do in
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