Guest Lucy Posted March 17, 2019 Report Share Posted March 17, 2019 Dear Sir or madam, I am using Mouse IL-4 ELISPOT basic kit for detection of IL-4 secretion from Mouse splenocytes. But I have encountered the problem that there were a lot of spots even from PBS injected mouse splenocytes without any stimulation. And the plate background is quite dark. (Please see the attached photo) I used 250000 cells per well. The cells were stimulated with 0, 0.5, or 2.5 µg/ml commercial influenza vaccine for 19 hours at 37°C. RPMI with 10% FBS was used for culturing. Diluted vaccine solution was added to the wells before splenocytes were added in and mixed. Protocol from MABTECH was strictly followed. The plate was washed with pre-packed PBS solution from Gibco. Streptavidin-HRP concentration was 1:1000. And 10-15 min was allowed for the TMB development before the plate was washed with diH2O and dried. CTL ImmunoSpot® Analyzer was used for image acquisition. Any suggestions to help me in reducing the number of spots from unstimulated wells? Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted March 17, 2019 Report Share Posted March 17, 2019 Interesting case. It is likely that the IL-4 spots are totally correct and that these are in vivo activated T-cells. They sure look correct. What haas been done with the mice prior to harvesting of the spleens? Any reasonable explanation that could explain why they would have in vivo activated T-cells in their spleen? best, Christian Link to comment Share on other sites More sharing options...
Guest Lucy Posted March 18, 2019 Report Share Posted March 18, 2019 Dear Christian, That's what makes me confusing: I did nothing other than injection of PBS to the mice (two individual experiment, two mice, newly opened pre-packed PBS). I cannot think of any reason why the cells looked activated. For your reference, I also attach the photo of IFN-gamma assay, where the same cells were used. In the same assay, I also have a mouse vaccinated 7 days ago and in vitro stimulated with 2.5 ug/ml vaccine for 19 hours.The IL-4 ELISpot photo was also attached. (Similarly, IFN-gamma assay of these cells responded normally.) Cheers, LU Lu Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted March 18, 2019 Report Share Posted March 18, 2019 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. Link to comment Share on other sites More sharing options...
Guest Lucy Posted March 18, 2019 Report Share Posted March 18, 2019 Dear Christian, I have some other plates with better PMA+Ionomycin stimulation results. I always use 250000 cells, same for IFN-gamma and IL-4 tests. Currently, I'm using only one batch of FBS (which I tested in deriving bone marrow dendritic cells). So I cannot help in ruling out the possibility of FBS causing dark background. I have not try PMA+Ionomycin on IL-4 ELISpot before, since my very first try of IL-4 ELISpot gave me a lot pf spots. But I shall include this as a positive control for any future assays. I'll contact the local distributor (http://www.houbio.com/) and talk to them about the case. Thank you very much for the help! I'll update once some progress are made. Cheers, LU Link to comment Share on other sites More sharing options...
Guest Mei Ling Posted March 18, 2019 Report Share Posted March 18, 2019 Dear Lu, Please contact any of our distributors below who supports both China and HongKong: NeoBioscience Technology Company (深圳欣博盛生物科技有限公司) Tel: 4006 800 892 (Hot line), +86 755-26755892(SZ), +86 10-88594029(BJ), +86 21-34613729(SH), +86 18024516375(GZ) Fax: +86 755-26755877(SZ), +86 10-88594549(BJ), +86 21-34613719 (SH) Website: www.nbs-bio.com Dakewe Biotech Co. (深圳市达科为生物技术有限公司) Tel: +86 10 64828744/54/84/94 (BJ), +852 2668 2848/5307 5566 (HK), +86 21 80166290/91/92/93 (SH) Fax: +86 10 64828012 (BJ), +852 8160 9290 (HK), +86 21 80166294 (SH) Website: www.dakewe.com Laizee Biotech CO., Ltd (上海莱兹生物科技有限公司) Tel: +86 10 6073 9963 (BJ), +86 21 6229 2399 (SH) Fax: +86 10 6073 9771 (BJ) Website: www.laizee.com Amyjet Scientific Inc. (武汉艾美捷) Tel: +86 (0)27 5962 6688 Fax: +86 (0)27 5971 6800 Website: www.amyjet.com Best regards, Mei Ling Link to comment Share on other sites More sharing options...
Guest Lucy Posted March 18, 2019 Report Share Posted March 18, 2019 Dear Mei Ling, Thanks, I'll try to contact Dakewe tomorrow in office hour! Cheers, LU 14 minutes ago, Guest Mei Ling said: Dear Lu, Please contact any of our distributors below who supports both China and HongKong: NeoBioscience Technology Company (深圳欣博盛生物科技有限公司) Tel: 4006 800 892 (Hot line), +86 755-26755892(SZ), +86 10-88594029(BJ), +86 21-34613729(SH), +86 18024516375(GZ) Fax: +86 755-26755877(SZ), +86 10-88594549(BJ), +86 21-34613719 (SH) Website: www.nbs-bio.com Dakewe Biotech Co. (深圳市达科为生物技术有限公司) Tel: +86 10 64828744/54/84/94 (BJ), +852 2668 2848/5307 5566 (HK), +86 21 80166290/91/92/93 (SH) Fax: +86 10 64828012 (BJ), +852 8160 9290 (HK), +86 21 80166294 (SH) Website: www.dakewe.com Laizee Biotech CO., Ltd (上海莱兹生物科技有限公司) Tel: +86 10 6073 9963 (BJ), +86 21 6229 2399 (SH) Fax: +86 10 6073 9771 (BJ) Website: www.laizee.com Amyjet Scientific Inc. (武汉艾美捷) Tel: +86 (0)27 5962 6688 Fax: +86 (0)27 5971 6800 Website: www.amyjet.com Best regards, Mei Ling Link to comment Share on other sites More sharing options...
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