Jump to content


  • Content count

  • Joined

  • Last visited

  • Days Won


Everything posted by Christian@mabtech.com

  1. No spots for IFN-gamma

    Ah, very good that we found the issue! Mabtech TMB substrate for ELISpot can be found here: https://www.mabtech.com/products/tmb-substrate-elispot_3651-10 Your strategy for the number of cells to incubate per well sound very reasonable.
  2. No spots for IFN-gamma

    Hold on, you are running an ELISpot basic kit! What substrate are you using? We have had experience before with people getting zero spots after trying to develop them using ELISA substrate. Will NOT work. The substrate need to be "precipitating" substrate, like BCIP/NBT Plus: https://www.mabtech.com/products/bcip-nbt-plus-substrate-elispot_3650-10
  3. No spots for IFN-gamma

    Dear Kenneth, in all liklihood your transfected cells are not secreting the IFNg cytokine. How many cells are you incubating per well? We occasionally run transfected cells in ELISpot when looking at exotic animal IFNg like Salmon for example. We get massive amount of secretion so we have to keep incubation very short. Only about 2-4h. Rather few cells per well also, around 500-1000. Otherwise the whole membrane is full of spots.
  4. Antigen-specific B cell ELISPOT

    The concentration for the biotinylated antigen should be optimized in trial experiments through a titration. I would recommend something like: 0,1ug/ml 0,5ug/ml 1,0 ug/ml Good luck Akira! Christian
  5. Antigen-specific B cell ELISPOT

    Dear Akira, results seem to have worked well with you coated antigen and detection of antigen specifc IgM! Nice! However, like you point out, in your second experiement using a biotinylated antigen, the first approach will not work since this setup is based on streptavidin-ALP in the detection phase. You will "short-circuit" the assay and end up with totally dark wells if you try it. Its a no go. Instead, I would suggest you do the so called reverse B-cell ELISpot! Here you coat the membrane with anti-IgM capture antibodies (MT11/12). You then add your cells containing the B-cells. During incubation all IgM antibodies secreted will get captured. After incubation you wash away the cells using PBS. Then you add your biotinyalated detection antigen for 2h. This will get captured only by the captued IgM specific for your antigen. You wash and visualize these spots using SA-ALP and BCIP/NBT PLus as normal. The results is often better looking and more senstive assay compared to when coating with the antigen itself. In this paper they show some comparisons: https://pubmed.ncbi.nlm.nih.gov/23454005/
  6. ELISpot cell dulitions

    It is smart to begin your project doing some titrations to figure out an optimal cell number for your particular antigen. Maybe responses are much better at 400K cell/well compared to 250K/cells. This is difficult to know prior to actually testing. However, once you have found a cell number where you dont feel limited by a lack of "cell-to-cell" contact, you can stick with that cell number for an entire study. ELISpot and FluoroSpot readers typically have good linearity on counting spots from 0-600 spots. With Mabtech IRIS we see good linearity between 0-2000 spots even beyond. As a result, you are not limited by the read out equipement for capturing both weak and very strong responders.
  7. ELISpot system suitability

    In all situations when you are interested in analyzing cytokine secretion by immune cells. ELISpot/FluoroSpot are reliable and recognized as one of the most sensitive assays among immunological methods.
  8. Antigen-specific B cell ELISPOT

    We are happy to help. Comeback any time Akira! best, Christian
  9. Antigen-specific B cell ELISPOT

    Dear Akira, Sorry we missed you post! For Human IgM we recommend the following setup: Capture mAbs (MT11/12) at 15ug/ml coating concentration (Code 3880-3-1000) Biotinylated detection mAb (MT22) at 1ug/ml detection (Code 3880-6-1000) And yes, the MT22 detection antibody works fine in Total IgM detection. best, Christian
  10. Mr

    Dear Hari B, Welcome to the forum. Here are my intital reactions/questions: 1. After 5 days stimulation with R848+IL-2, the amount of IgG in the cell cultures is massive. Washing 1x is on the small side. I would recommend doing 3x or 2x and carefully removing all supernatant all the way down to the cell pellet. In this way, free IgG in solution will be reduced which can ofcourse cause high backgorund. 2. The 200,000 cells/well for Total IgG is too high. A better option would be 50,000 cells/well. For your antigen specific wells 200,000 cells/well is ofcourse good. 3. When coating with only PBS, do you include a blocking step in your assay with cell culture medium containing 10% FBS? Blocking of the PVDF membrane is needed. 4. The well names of you uploaded images is not shown to me. My guess is that the really dark ELISpot membrane above is the PBS control, correct?
  11. Using Tween20 detergent in Wash Steps of ELISpot

    Dear Del, You have made an important observation! Yes you are right, Mabtech does not recommend Tween and the reason for this is simple: in our hands, with our protocol, it leads to darker ELISpot membranes. There is absolutely no benefit, both for precoated kits and the basic kits where you coat yourself and follow our recommendations on EtOH preactivation! At the same time, other ELISpot practitioners are adamant on the use of Tween as a critical component in making ELISpot results look better. How can our viewpoints be so different? Well, it all comes back to how the plates are set up. We always recommend that you should EtOH treat your plates prior to coating. This increases the binding capacity of the PVDF membrane, and combined with a good amount of capture antibody (1-1.5 ug/well), will always produce better results compared to NOT using EtOH pre-treatment. In a situation where you follow Mabtech's recommendations, Tween provides no benefit and will actually "hurt" your results by making membranes noticeably darker after the plate has been developed. This is true for both the precoated and the freshcoated, but even more noticeable in the latter. However, in cases where you coat your plates yourself and decide not to perform the EtOH pre-treatment, results will actually benefit from the use of Tween. Spots will appear more clear and the "dark-membrane effect" seises to exist, or atleast becomes much less noticeable. It would therefore appear that the use of EtOH fundamentally changes the effect induced by Tween on PVDF membrane. Spots simply look better in my opinion. At the same time, excluding the EtOH activation will make your ELISpot assay suboptimal in terms of sensivitiy. If you buy percoated, plates have been made under optimal conditions so there is no need to use tween.
  12. Background Problem with Mouse IFN-gamma ELISPOT assay

    Dear Silvia Pérez! Welcome to the forum. I have looked at your images and made some comments about it in the attached PPT. Please have a look! Overal I think this could be a case of a particular stimuli causing darkening of the membrane. Some peptides induce this effect. I think you could reduce the problem quite a alot by turning off the substrate reaction earlier. Most people tend to overdevelop their plates. Your plate is still wet as can be seen in the photos. Dry it completely and you will see that the membrane does "lighten". You will probably still be able to make out the spots from the dark membrane background. Feedback.pptx
  13. Hydrated too many ELISpot PLUS plates

    Hello David! I have personally kept pre-coated plates like this for 48h and then done the assay without any issues. Longer storage times could be theoretically possible but that is nothing we have evaluated ourselves. Furthermore, please keep in mind: - Some bacteria like to break down antibodies. If such a contamination have made it into one of the wells by help of the cell culture medium, this could end bad for the capture antibodies, drastically affecting the assay. - With cell culture medium in all wells you still have evaporation when stored in 8C. Could be a good idea to wrap the edges of the lid with parafilm in order to minimise this effect.
  14. Plate coating

    Hey Sarah, Yes, I have done this many times without any problems. One thing to consider is that the antibody coating solution inside of the wells will start to evaporate even when kept in a cold room with the lid on. So one can wrap the plate in Parafilm to minimize evaporation. Otherwise all liquid could evaporate and leave the membrane exposed to air. Not good. Also, any contaminant that entered into the plate during the plate coating process would get extra time to "grow". So be sure to etoh activate and add capture antibody in sterile conditions. But that is a given ofcourse.
  15. LOW signal with plasma samples

    Dear Sanda, How does you standard curve look like? In normal healthy donors you typically have very low levels of IL-10, IL-6, TNFa, IFNg and IL-1beta in plasma. By contrast, in inflammatory diseseas like sepsis for example you can often get above 1000pg IL-6 and IL-10. /Christian
  16. technical support B-cell IgM

    Dear Michelle, Welcome to our forum! When it comes to IgM I really believe that all spots you are seeing in unstimulated controls are "real" spots. They are not artefacts. However, there is a clear stimulation increase and we want to set our counting paramters in a way so that this increase in the number of bigger spots is accurtaly captured. For me this is about making a research decision. Pick a count setting and then stick to it throughout your study, given that all donors and plates have been handeled in the same way! Evalute 3-5 donors before making a decision on which setting is good. If plates have not been developed equal times with substrate having the same count-setting could be impossible. Excluding "real" spots like this is in my mind totally fine, although I prefer If the "thresholding" is done in a consisten manner on all plates due to the study having been performed in the same way over and over again (substrate development time being crucial). The important part is that you should be able to send your plates to a collaborator in United States with the same type of reader. He should then be able to use the same count settings and extract the same spot counts from your plates within +-3%. I have taken your PPT and shown with arrow which kind of spots I would want to count in your unstimulated controls. I have done this pretty quickly so dont take it litteraly. But you can see that I would want you to use a count setting threshold which focuses on counting the bigger spots. Exclude the small unspecific background. In your example you will get a very nice stimulation index. Does all your IgM ELISpot plates look this good? Great job. IgM feedback.pptx
  17. Antibody coating

    Hi Emma, It certainly works doing a quicker coating protocol of 2h in 37C. However, we have never done proper studies at Mabtech comparing our standard protocol (4C overnight) with that of 2h in an incubator. It is possible that the sensitivity of the assay would go down slighly, but that is a pure speculation. My gut feeling is that the difference is very very small. But this you will have to validate yourself.
  18. Stimuli for ELISPOT

    No problem So I personally prefer using fresh isolated cells since this pretty much guarantees good viability. Cryopreserved pbmc can have excellent viability (if frozen and thawed correctly), but in my experience rather few labs have this perfectly implemented. Viability is bad and the ELISpot result will be suboptimal. Nevertheless, if you have confidence in the viability of you cryopreserved cells, go with that. Works fine. This is what we use every day at Mabtech. It lowers the workload. Final recommendation: dont overdo it in your first experiment. Dont include 8 donors in one single go because it just increases the liklihood of something being pipetted incorrectly. Start of small and build up complexity! Good luck!
  19. Stimuli for ELISPOT

    Hey! 1. If this refers to the incubation of the cells within the actual ELISpot plate, you do not need to supplement with recombinant IL-2 in the cell culture medium. Should not be necessary. We use standard RPMI 1640 supplemented with 10% heat-inactivated FCS, 1 mM glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 0.5 mM HEPES. However, you can also use serum free media and then I recommend AIM-V. 2. I strongly recommend you use 10% heat-inactivated FCS. Pooled human serum is bad idea since it can contain heterophilic antibodies which cause background in the ELISpot membrane. 3. ELISpot have historically not needed anti-CD28 but I think one can include in a few wells and compared it not using. On the other hand, this increases complexity of the experiement and raises the liklihood of making misstakes. So skip it in the first experiment and see how it goes! best, Christian
  20. SDS and environmental hazards

    The Fluorescence enhancer contains a very low concentration of Kathon CG at 0.002% concentration. It is listed in the SDS for the actual kit you purchased. We recommend it is diposed according to local regulations. In most labs I have been in this low concentration is not deemed dangerous in any way and is just flushed down the drain. Other than that the enhancer does not contain anything "dangerous" to the environment.
  21. Stimuli for ELISPOT

    Very good! We are here to help! When you buy the kit we include a positive control that is anti-human CD3. A very reliable polyclonal activator of T-cells. Recommended cell number for this is 50,000-100,000 cells/well when analyzing human IFNgamma. So a littlebit less compared to antigen specific wells that typically need 250,000-400,000 cells/well. It is good to always include a few wells with a postivie control since it will tell you that the cells are in working order. Good luck!
  22. Stimuli for ELISPOT

    Peptide stimulation typically works very well in ELISpot. This is what most vaccine papers using this method employ. Given that the peptide induces a response it is very reliable. In every quality control at Mabtech we include the CEF peptide pool. As long as the donor is a responding one, it always works. One should make sure that the cells are of good quality. Viability should be over 84% and this includes both dead and apoptotic cells. By contrast, some researchers are investigating whole proteins. These need to go through much more extensive processing and presentation and here there can be limitations in getting the T-cells to respond in certain scenarios. One might need to go up very high in cell numbers/well and one might need to supplement with anti-CD28 antibody stimulation to see a clear response. Some go to even greater extent and load autologoous dendritic cells with antigen and add those in great numbers to isolated T-cells. Or in the case you are refering to: mix in cells infected with virus expressing the antigen. All of this is ofcourse possible but one should always start with the easy approach: Take your peptide and run an ELISpot at 250,000 cells/well and 400,000 cells/well. Titrate the peptide so you know you get optimum stimulatino. Usually stimulation is effective at 2ug/ml but go up to 4-5 just to be sure. Make sure your cells are in good condition. Get a precoated Mabtech IFNg ELISpot kit. Select an ALP kit like this one: https://www.mabtech.com/products/human-ifn-gamma-elispot-pro-kit-alp_3420-2apt-2 For your initial testing don't coat yourself. Most often when ELISpot goes wrong it is when you make mistake in coating. Precoated kits from Mabtech have been done under optimal conditions which include the etoh activation. Performance is much much better compared to coating yourself but not doing the etoh.
  23. Minimum cell number

    T-cell analysis in ELISpot/FluoroSpot is to a certain extent dependent on cell-to-cell contact, even for some polyclonal stimuli. PHA works fine at 50,000 pbmc/well for IFNg, but can drop to almost nothing at 20,000-25,000 PBMC/well. This can vary from donor to donor but it is important to understand that it is not totally linear. The same is true for the anti-human CD3 stimuli from Mabtech. It can induce IFNg very reliably at 50,000 cells/well but below 25,000 you suddenly go off a cliff. By contrast, PMA+Ionomycin is much more independent of cell-to-cell contact and will stimulate T-cells great even at very low numbers of cells/well. I think I have seen people use just 5000 PBMC per well and it worked. PMA+inomycin can be tricky since it is a light sensitive stimuli and I recommend to always prepare it fresh before use. best, Christian
  24. IFN-Gamma ELISPOT No Development?

    Sorry for not getting back earlier Eva. I am out traveling but have looked at your response from time to time and here is what I have to say: - You certainly have spots in the last uploaded elispot images. I dont have any magical recommendations for you that can explain your sudden lack of spots in some plates. But If I were to speculate it would be that the cells are not in good condition. I say this because if it was an issue with reagents not being added correctly, the first uploaded plates would be completely blank, but they are not. If I look carefully I can see wells that seem to have real spots in them. - So one scenario could be that there has been a mistake in dilution for example. Instead of adding 100,000 cells/well, only 10,000 PBMCs are added. They result is no response and PHA postivie control can suffer a lot as well due to it needing a certain level of cell to cell contact to work well. - Another thing could be viability of the cells. If thawed PBMC are handled improperly, for example they are left for long periods in freeze medium before being transferred and washed by cell culture medium, quality of the cells can go down fast. Say for example you thaw 10 tubes of cells from one donor simultansouly. Tube 1 only gets 1min of freeze medium treatment after being thawed in a water batch, but tube 10 will get 8min. That can have a big impact.
  25. IFN-Gamma ELISPOT No Development?

    A few comments/suggestions from my side: - In the 3 plates you have uploaded I do not see any positive control wells full of spots. Is this just a co-incidence? Or is it always like that? In general, when using a positive control like anti-Cd3 or PHA you get above 500-1000 spots/well atleast at 100,000 cells/well. It is good always having a few positive control wells in each plate to assure that cells and the assay is working well. - When I coat ELISpot plates myself I always do EtOH activation prior to adding capture antibody. I then use the recommended concentration of 1,5ug/well. In you example you say you add 5microliters of antibody to 10ml of PBS and then coat with 100ul of that solution. I would use 150ul as a comparison. You essentially use 0,05ug/well of capture antibody, which is too low. You could go down to 0,5ug/well but you would then need to add 50 microliters of mab to 10ml PBS when preparing your stock. This could explain a total lack of spots in general. - I think it would be good if you tested pre-coated plates in parallell to just get a feeling for how it could look when optimally prepared. Pre-coated is more expensive but it ensures an optimal result. You spend a tremendous amount of money and energy on generating this type of data so for me it makes little sense saving a few bucks on the final read out. Our ELISpot PRO kits come with one-step detection where 7b6 is directly conjugated to ALP: https://www.mabtech.com/products/human-ifn-gamma-elispot-pro-kit-alp_3420-2apt-10 In this kit we also include our own BCIP/NBT Plus substrate. I have never seen the BIORAD substrate before. A substrate not optimized for ELISpot can have devastating effects. Is this BIORAD substrate actually a precipitating substrate? It needs to be otherwise you wont get any spots.