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tylsan

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    Thanks for all your insight and tips Christian and Mabtech! It's really appreciated!
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    One final thing, if I'm going to use a biontinylated antigen, would I still be able to detect both IgA and IgG antigen specific cells in the same well? (See attached image for clarification). So I could follow the same protocol coating with capture anti IgA and IgG, and then when it comes time to detect cells, I could incubate with biotin-antigen and detection anti-IgG/IgA? I would not need to use 16 wells per sample either, but only 12 since I would not need antigen only wells if I am thinking correctly? I would be able to detect total IgA, total IgG and antigen specific IgG and IgA in the same well theoretically since the antigen specific spots would be two colored correct? Sort of how Peter did with identifying antigen specific IgG subclasses in the article you mentioned earlier. Or would it be better to have separate wells for detecting IgG and IgA. In that case would a normal ELISPOT be better here since I would only need to detect one type of spot? Or am I making this way too complicated? /Tyler
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    Ok I suspected that the membrane drying could be an issue. I'll just have to try to be quicker then next time Thanks for all your help! Really appreciate it! /Tyler
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    Hej Christian! Noted! Thanks! I actually did this. I did a two day stimulation with the cells (I'll try 3 days next time) and finished developing that plate today. There were a lot more spots in the positive controls, but still sadly nothing with the antigen specific wells. Thanks for the tip! I'll give it a try. One more question, could the fluorescence enhancer cause the high background in some of the wells? Or other thoughts on the high background? I saw your YouTube video and really tried to make sure to get rid of the excess. Thanks for all the help! /Tyler
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    Hi Christian! Thanks for the speedy resonse! Thanks! I was happy to see at least part of it worked Sorry about the low resolution. This is just the screen capture from the AID software. Sorry I realize I could have been clearer there. They are two different donors. I used a separate 96 well plate to make the dilution series for the samples so that I could transfer over them over easily. Yes after resting 1h, the cells were washed and resuspended and then transferred to my dilution plate before plating on the fluorospot plate. The lack of Ag specific spots was disappointing. We see a significant increase in plasmablasts after vaccination with flow cytometry on the fresh cells and assume they are vaccine specific so we do expect to see some. The antigen is from the vaccine. It's a live attenuated virus that was grown and inactivated using Triton X-100 (0.5%), KCl (0.6M) buffer and heat inactivation. So the whole virus is still present and we hoped it would work for coating. Could a whole virus particle be too large to bind? I talked with Peter at the SSI meeting last fall (if I remember correctly) and discussed doing a reverse FluoroSpot, but were unsure how to go about tagging our antigen since the antigen is a whole virus. Any ideas on tagging viral particles? Since we (right now) only have frozen cells we could enrich them using this? https://www.stemcell.com/products/easysep-human-pan-b-cell-enrichment-kit.html We were hoping to do these experiments on fresh cells, but the collection started before we had unfortunately fully optimized this assay. I also have an FluroSpot incubating now from the same donors, but instead I stimulated these cells for 48 hours. We'll see how that turns out this afternoon. Yes the whole plate received 35% Ethanol pre-treatment for approx. 30 seconds. Thanks so much for your help! /Tyler
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    Hi Mabtech! I used your human IgG/IgA/IgM FluoroSpot kit (FS-050617) to look at total and vaccine specific plasmablasts after vaccination with different licensed vaccines. Overall the kit and protocol was well constructed and easy to follow! I used cryopreserved cells that I rested for 1 hour before moving them to the plates for incubation. The plates were coated with either the anti-IgG/A/M included in the kit or 10 µg/ml of inactivated whole virus (specific to the vaccine). The antigen specific wells didn't seem to work at all, but the highest concentration of cells worked well with the total IgG/A/M and are similar to our FACs data from these time points. However in certain wells there is really high background. I used a 12-channel multichannel pipette so I would expect that all the wells in the row to look somewhat similar, but they don't seem to be that way. Any ideas why this could be? Thanks! Fluorospot IgG:IgA:IgM.pdf
  7. Hi Christian, 1. Thanks for looking into this for me! I'll stick to the PVDF membrane plates! 2. For my antigen specific wells I coated with both 500 000 cells and 180 000 cells. These cells were stimulated for 5 days with PWM, SAC, CpG and IL-10. I was also considering taking a portion of the cells before stimulation and look at ASCs as well after they've rested for an hour after thawing. /Tyler
  8. Thanks for the speedy response Christian! I have tested the same protein biotinylated and see no spots either unfortunately. I am starting to suspect the antigen we are using may be the issue as I am getting consistent IgG+ wells. I will have to investigate further. But just to confirm that I understood your response about using a pre-coated ELISA plate, even if it's a working ELISA kit for our same serum samples, there simply won't be as much antigen available for the specific antibodies to bind to? Thanks again! /Tyler
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