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Christian@mabtech.com

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Christian@mabtech.com last won the day on January 8

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About Christian@mabtech.com

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    Accuratly decribe our official policies and recommendations
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  1. 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
  2. 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.
  3. 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!
  4. 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
  5. 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.
  6. 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!
  7. 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.
  8. 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
  9. 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.
  10. 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.
  11. B-cell ELISpot IgG dark background staining

    Hello there Michelle! The background does look very high. A few questions/comments: 1. Did you pre-activate the B-cells for 3 days together with R848+IL2 in order to get the memory B-cells going? I will pressume yes but just checking. 2. Prior to adding the cells to the ELISpot plate, did you wash them twice? There is a crazy amount of IgG in the supernatant of these precultures and it is important to wash away all IgG, otherwise they will give a general background to the membrane. Washing 3x can be smart. 3. Did you use tween anywhere in the assay? If you used the general ELISA wash during the detection phase of the ELISpot, it is easy to forget that ELISA wash do in fact contain tween. Tween gives weird backgrounds if you etoh treat your plates. No benefit. 4. Do the images generated in the reader match what you see with your own eyes? Maybe a strange question, but something is seriously weird in the images you are providing. There seems to be some optical effect in many of the wells. Like there is dust in the lens of the reader or something? Maybe the bad background only exist in the reader?
  12. Stimuli for Elispot assay

    Which peptides you use in an ELISpot study is something the principal investigator of your research study must decide. We cannot do this for you. It all comes down what sort of scientific question you want to answer in your study. What is it in the T-cell immune response of Liver cancer patients that you want to investigate? Maybe you have a hypothesis that you want to evaluate?
  13. Stimuli for Elispot assay

    Dear Heidio, If you want a positive control peptide, the CEF peptide pool can be a good choice. This contains CD8 speicific peptides from virus infections like CMV, influenza and Epstein Barr. Most donors respond and you can get a more functional positive control
  14. many spots in negative control

    Dear Janou, Welcome to the forum. I have a some feedback: IFNg elispot does not always result in a very low background (0-5 spots/well). If you run 50 healthy donors you will probably have 90% very low back background, 5% moderate background and 5% high background. With 250,000 cells/well, I would say moderate background is 10-15 spots/well and high background is 25-35 spots/well. Factors such as isolation technique, cell culture medium, serum used and the sensitivity of your ELISpot assay will all affect these numbers. Furthermore, the count settings of you ELISpot readers is greatly, greatly influential. Why do some totally healthy donors have a lot of background spots? I do not know for sure, but my speculation is that the "immune status" naturally plays a role in these background numbers. Some might be fighting off a "spur" of CMV infection somewhere in the body. This happens all the time (and without any symptoms) and could in theory generate a higher background of IFNg in unstimulated PBMC. But there could also be a factor of outside stimulation like isolation technique and bad serum. Patients will naturally have a much more complicated "immune status" compared to healthy donors, so it is not surprising that background levels tend to go up. Furthermore, one should not forget that NK-cells also have the capacity to secrete IFNg. Maybe these cells are more activated in the blood stream of sarcoïdosis patients, which could be an alternative explanation to the background observed. Looking at you wells it does look like well F6 in the second plate has a lower number of spots compared to the unstimulated F2. I would here say two things: - In F6 you have incubated PBMC with antigen. Although no antigen specific T-cells have been activated, the T-cells could still be affected differently by the antigen added. Monocytes could take up the antigen, process it and release cytokines which fundamentally changes the cytokine environment of the well. We know from experience that many antigens are contaminatied with TLR-ligands. These monocyte derived cytokines could in turn downregulate the T-cells/NK-cells secreting IFNg directly exvivo. This could be one explanation for the phenomenon observered. - You reader says there are 46 spots in well F6 and 90 spots in the unstimulated control F2. These numbers will naturally be very dependent on the settings of your AID reader. Maybe you have the count settings rather high, meaning you only count rather dark and bigger spots? In well F6 you might in reality have 80 spots, but only 46 are counted by your settings due the high threshold you have applied. Something to look into. Take the F row as an example, here I would say that only well F6 falls below what is considered natural variability for an ELISpot assay. With a different count setting the difference could even be smaller? We have made a tutorial on the AID count settings on youtube. Can be found here: Or even better, consider switching to our next generation IRIS reader. With this reader I really recommend switching over to FluoroSpot, even for single color IFNg analysis. The RAW technology makes life so much easier and the RSV values are increadibly fun to analyze:
  15. Mouse IL-4 ELISpot

    What is the source of your FBS? This could be the source of the very dark membrane you are seeing. Do you have another source of FBS that you could run side by side maybe? When seeing your IFNg results I am very struck by the lack of spots of IFNg in the positive control wells (PMA+ionomycin). This is very unusual and indicates that the cells are not in great condition or being very badly inhibited by something. How many cells/well did you incubate here? Do you have a postive control well for mouse IL-4 as well (PMA+ionomycin)? I have checked our QC experiment for mouse IL-4. We use thawed mouse spleen cells and backgrounds are zero. Attach the photo. You know what, we should also exclude the coating process as being a possible culprit. I would be happy to send one of our distributors a pre-coated mouse IL-4 plate for you to run side by side (free of charge). Just contact a distribtor and show them the link. We could arrange that.
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