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Christian@mabtech.com last won the day on April 22

Christian@mabtech.com had the most liked content!

About Christian@mabtech.com

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    Mabtech representative

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    Accuratly decribe our official policies and recommendations
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    Stockholm, Sweden

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  1. 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.
  2. 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.
  3. Antigen-specific B cell ELISPOT

    We are happy to help. Comeback any time Akira! best, Christian
  4. 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
  5. 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?
  6. 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.
  7. 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
  8. 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.
  9. 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.
  10. 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
  11. 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
  12. 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.
  13. 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!
  14. 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
  15. 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.