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  2. 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
  3. technical support B-cell IgM

    We are trying to set up a B-cell Elispot assay for IgM. but are facing some new challenge, deciding which spots are legit and which are false, setting our protocol boundaries spots size wise. We were hoping someone could help with that decision. For us it is unclear what size spots you can expect for IgM. But also the differences in spot size in unstimulated and stimulated wells is notable. Does one of you have a answer? Thanks! IgM Elispot.pptx
  4. Antibody coating

    Hi Emma, Yes, I agree with Christian and it is possible to adjust the protocol a bit (but as Christian writes we have not done comparison studies). I sometimes coat in RT (for 3hrs) and block overnight instead, if that suits my experiment schedule better. If you are tight on time, you could instead go for the ELISAPRO kits, with precoated plates. That will save you both time and decrease variability. Link to ELISAPRO IL6: https://www.mabtech.com/products/human-il-6-elisa-pro-kit_3460-1hp-2 Link to ELISAPRO IFNalpha: https://www.mabtech.com/products/human-ifn-alpha-elisa-pro-kit_3425-1hp-2 Good luck! Lena
  5. Antibody coating

    Hi Christian, that’s very helpful, thanks for your quick response! Emma
  6. 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.
  7. Antibody coating

    Hello, I am using two of the MabTech ELISA development kits (IL-6 and IFNa HRP), following the instructions provided which have been working well. I'm tight on time this week and want to know if it's possible to change the overnight antibody coating step to save time - I've read that with some assays you can incubate at 37C for 2 hours instead. Please can someone let me know as soon as possible if this would be suitable to do with the kits? Or what the minimum time for coating at 4C is? Thanks! Emma
  8. Stimuli for ELISPOT

    Hey, Thanks a lot. It is really nice hearing from you on this. If I have any other doubts, I'll write you. Regards, Alok
  9. 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!
  10. Stimuli for ELISPOT

    Well said! Really good suggestions before starting off with my experiment. One more thing I would like to ask: Should I use cryopreserved PBMCs or freshly isolated PBMCs. Its bewildering hold up, as my neighbouring labs in the institute tell me to use freshly isolated ones, which might increase complexity as how many samples I get in a day and freeze thawing of reagents and materials (which may affect their efficiency). Please suggest on this. Sorry to bother you again!
  11. 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
  12. Stimuli for ELISPOT

    Dear Christian, My peptides have arrived and my Mabtech ELISPOT kit will arrive shortly. I have some technical doubts. 1. During storage of PBMC population, do I need to add recombinant human Interleukin - 2 ? If yes, should I use 5U/ml or 20U/ml along with 100 U/ml penicillin and 100mg/ml streptomycin? 2. While setting up my ELISPOT assay, should I use 5% pooled Human serum or 10 % FCS in RPMI 1640 ? 3. Do I need to use anti CD-28 additionally during EISPOT assay? Thanking you in advance.
  13. 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.
  14. SDS and environmental hazards

    Hello, How should the fluorescence enhancer fluid be disposed of. Are there any components that are toxic to the users or the environment. A search for SDS or MSDS yields not results. Thanks M
  15. 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!
  16. Minimum cell number

    Thank you so much for the reply, then there is hope- I'll give it a try and evaluate using different stimulus. Do you by the way have any advice on how to only use parts of the plate for a pilot experiment? Can this even be done considering that the whole plate is put into the incubator? Best, Emelie
  17. Stimuli for ELISPOT

    Dear Christian, Thank you for your valuable suggestions. They will be helpful when I will start my assay. I understand that cell quality, number of cells and quantity of antigenic stimuli I quite important for expecting spots. Now I understand why there is so much variation among research papers publishing their data and protocol. Also, it is really kind of you suggesting which ELISPOT kit I should buy for my assay.
  18. 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.
  19. Stimuli for ELISPOT

    I want to do ELISPOT for IFN gamma to assess T cell memory response against HPV 16 E6 peptides. I have a doubt about stimulating cells during ELISPOT. Monocytes are cells which function in presenting antigen to T cells and they will be present in PBMC population. We expect monocytes to take up the antigenic peptide and present it to T cells in context of respective MHC I/II molecule. However, some papers recommend isolation T cell clones infected with Vaccinica virus expressing HPV 16 E6 protein. Do you have any suggestions for this? Also can you cite a paper where Mabtech ELISPOT kit has been used which may help in this.
  20. 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
  21. Minimum cell number

    Hi Mabtech, I was wondering if you have any experience on the minimum cell number to use for an IFNg/IL-17 flurospot assay? I am planning to sort cells and will have very limited cell numbers (in the single thousands range). I plan to stimulate the cells in various ways (all polyclonal stimulation) and it would be very useful with some input if this approach is even feasible in the context of fluorospot? Thanks for your always very helpful insights, Emelie
  22. 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.
  23. IFN-Gamma ELISPOT No Development?

    also, the PHA is well H12. thanks!
  24. IFN-Gamma ELISPOT No Development?

    hi Christian Thanks for getting back to me. Our positive control was PHA and usually we get nice development where we see easily countable spots and our PHA wells are blown out from so much expression of IFN-gamma due to activation. We always have a positive control. The problem in this case was that non of the controls showed up because the entire plate was stained purple. I've run about 200 ELISPOT plates using this exact protocol every time and my lab has run at least 300 with this protocol. We've never had a problem like this. We have spent months optimizing this assay (with and without pre-coating, with and without EtOH, different titrations of Ab to use) and this is the combination that works the best for us--we are looking at IFN-gamma expression in PBMCs from people living with HIV who are ARV-suppressed's response to HIV peptides. Any other combinations have yielded too much background. I also ran this set of plates batched with another set and the other set developed perfectly. We've also used the biorad substrate for years and have never had a problem. As far as I know, it is a precipitating substrate. I've attached pictures of one of the plates that we ran batched that did develop. Thanks!
  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.
  26. IFN-Gamma ELISPOT No Development?

    Here's a picture of a plate from the last time this happened (not yesterday's plates, those are still sitting in 1X PBS in case I can rescue them). Thanks! Best, Eva
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