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  1. No spots with human perforin ELISpot kit

    Hi Jens and Christian, Thanks for your very detailed response with lots of info and some good points. Here are answers to your questions: • I have in the past had very good results with the perforin ELISPot testing PMA/Ionomycin stim of sorted subset of cells. I only had 5000 cells per well then and stimulated 24 hrs which worked really well. That was when I was located in different lab though and I cannot remember which plates we used, but pretty sure they were PVDF. • We know that the PMA+Ionomycin is working. We only purchased it a few weeks ago (actually just before the ELISPOT reagents arrived) and IFNg comes up positive in ELISAs and we detected IFNg, TNF and CD107 by FACS after PMA/Ion stimulation. We have some anti-CD3 and anti-CD28 here that we could try but I don’t think that would make a difference since I know our stock of PMA/Ion is new and working well. • We used the coating antibody at the recommended concentration (30ug/ml). • We are coating the plates ourselves because we wanted to use this on sorted cells again and would not use a whole plate at a time, thus it would be wasted with pre-coated plates. • When we pre-wetted the plate with EtOH (test 3) the wells went gray and we left it for no more than 1 min as the protocol suggested. • Potentially the substrate is not ideal although would you not expect to see something (faint spots maybe?). Anyway, I will try the BCIP/NBT-plus. • We use frozen PBMCs and we do cell counts with trypan blue and we consistently have very good cell viability around 90% and above. Later on for sorted cells these will be sorted in presence of a live/dead stain so that only viable cells are sorted. • I agree that I think the issues lie in the coating (and the plates we are using may not be ideal) and also in the substrate. I think it would be worthwhile testing a pre-coated plate side by side with one of our plates. Regards, E
  2. No spots with human perforin ELISpot kit

    Hi Jens, We have now tried this ELISPOT three times with slight changes from each, but still has not been able to see any spots. I’m attaching a photo for your reference. This is the protocol we used for each round: First test: · Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate. · pre-wet with PBS 200ul. · blocking with RPMI1640 supplemented with 10% FCS. · Stimulation PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control. · Cell concentrations tested: 50 000, 10 000 and 5000. · All washing and antibody solutions according to the protocol. · Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) (Sera Care Cat # 5420-0030(50-81-00). · Development 30 min. Second test: · Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate. · pre-wet with PBS 200ul. · blocking with RPMI1640 supplemented with 10% FCS. · Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control. · Cell concentrations tested: 50 000, 10 000 and 5000. · All washing and antibody solutions according to the protocol. · Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week) (Sera Care Cat # 5420-0030(50-81-00). · Development 60 min. Third test: · Hydrophobic high protein binding immobile-P membrane (Millipore MSIPS4510) plates. · pre-wet with 35% EtOH 15ul. · blocking with RPMI1640 supplemented with 10% FCS. · Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control. · Cell concentrations tested: 100 000, 50 000 and 10 000. · All washing and antibody solutions according to the protocol. · Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week). · Development 60 min. Hope you can give us some pointers of how we can make this ELISPOT work.
  3. No spots with human perforin ELISpot kit

    Hi Mabtech, I have tried to set up this ELISPot assay for human perforin, but have been unsuccessful. I have been using PMA/Ionomycin stimulated PBMCs (stimulated for 24 hrs which I have done in the past) but have not been able to detect any spots. We are using Multiscreen HA plates (mixed cellulose Ester Membrane plates) which is commonly used in our lab for other ELISpot assays. Otherwise we follow the protocol precisely. Can you please some ideas of what might be the reason for lack of spots? Regards, E
  4. How can I wash adherent cells completely?

    Dear Mabtech, Recently, I bought IFN-g ELISPOT kit pro and tried to find condition. I used PBMC and adherent cells, after 24 hours, I did assays on manufactured protocol. But I couldn’t wash out cells completely and I couldn’t detect signals (positive control is working). Then, I’m thinking to use Tween-PBS for the wash buffer, but your company doesn’t recommend it, I know. How can I solve this problem? Can I use accutase or Trypsine enzyme for a while? I am attaching images from the wells. Adherent_cells.pptx Regards, T
  5. Maximum sensitivity in ELISpot?

    Hi Mabtech, In terms of sensitivity, I see that the ELISpot assay can detect as little as one spot. In your experience, if there was only one antigen reactive T cell in 600,000 PMBC plated and you pulsed the well with presented antigen (for that one T cell), would that T cell secrete enough IFNg over time in order to be detected in this system? I am looking for a needle in a haystack, and I want to make sure this assay is an appropriate one. If it isn’t, can you recommend another product that would be sufficient? Regards, M
  6. Ethanol pre-treatment of strip-plate?

    Dear Mabtech, I purchased #3321-2A from you. For the PVDF plate, I use the type #M8IPS4510 from Millipore. I’m having problems with the pre-wetting step. I add 15ul 35% ethanol per well for 1min, but there are some white spots in the well. It seems like the membrane didn't get properly activated? I ‘m attaching a photo of the plate here: Have you seen this problem before? Is the problem the plate? Would you give some advice for it? Thank you! //C
  7. Is serum free medium ok in ELISpot?

    Hi Mabtech, I just purchased the Human IL-2 ELISpot BASIC (ALP), product code: 3445-2A, and have some questions about the data sheet (see attached image). 1. The medium in step B2, is it PBS or cell culture medium? 2. “The same serum” in step B2 is what kind of serum? 3. Does step B2 mean that the serum is added to the medium and the serum concentration is 10%? 4. I use serum free medium for cell culture, is that ok? //D
  8. What to expect from the mouse apoE ELISA?

    Hi Mabtech, What apoE values should we expect in WT mice? Best M
  9. Can I use the same ELISA plate washer for FluoroSpot?

    Hi Mabtech, I am currently running Flurospot plates from Mabtech. I have a general question when it comes to washing plates for the Flurospot: Would you recommend to always use separate plate-washers for ELISA plates and Flurospot plates or would you say that it is overall ok to wash both ELISA plates and Flurospot plates with the same plate washer? Thanks a lot in advance for your help Best Sabrina
  10. White spots

    Hi Jens, Thanks for your help. Yes, I used Tween for the washing steps. Will stop doing that from now on. //C
  11. White spots

    Hi Mabtech, I’ve used your mouse IFNg ELISpotBASIC kit (#3321-2A). The result is attached. Why are there some white spots in the well? With regards, C
  12. ELISA kit to measure anti-IgE antibodies

    Hi Mabtech, I have a particular request: I am looking for an ELISA kit able to measure human antibodies against IgE (a sort of "auto-antibodies”). Have you any kit or suggestion for this purpose? Regards, L
  13. How to dry the plates?

    Hi Mabtech! I was wondering if you could suggest the best way to dry the fluorospot plates. Should they just be left at room temp to dry or can they go in an incubator for a while? Thank you in advance for your help. Best wishes, A
  14. Blank wells after development

    Hi Jens, Thank you for your feedback. Based on your results I agree with your evaluation that the well apparently did contain detection antibody, but that somewhere during the detection process something has been suboptimal. And, as you say, it is difficult to pinpoint the reason for this. Do you have any further recommendations for avoiding such white wells in the future? Do you recommend following your “recovery” protocol if we experience similar blank or almost blank wells? Regards, D
  15. Blank wells after development

    Hi Jens, Two of the plates are on their way to your head office in Sweden. Regards, D
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