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Background Problem with Mouse IFN-gamma ELISPOT assay

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Guest Pavel

Dear, 

I am using Mouse IFN-gamma ELISPOT basic kit for measuring IFN-gamma secretion from Mouse splenocytes. But I have background problem and i cannot get rid of them. I have got lot of true spots but the problem is background. Here is my conditions: I used 150000, 200000 and 250000 cells per wells. I used PHA-M as positive control (10ug/ml, 20ug/ml). Stimulation time was 24 hours in suitable condition. As culture media, I use RPMI with 10% FBS. After that I follow all the steps stated on the protocol from MABTECH. I used BCIP-NBT substrate. First I tried 20 minutes then I got back ground. Later in several times, I used 10, 15 minutes with the same condition. But The back ground was still there. I also used less concentration of substrate (30ul, 50ul, 75ul). But the back ground was there. 

Before stimulation, I thawed the splenocytes from -80C and put into 37C for more than 1 hour. I am really confused now. Is the problem arise from FBS or longer time stimulation? 

Please help me is this regard. 

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Dear Pavel, welcome to the forum!

General background staining of the membrane typically can be traced back to these problems:

- The use of Human AB serum in the cell culture medium. Human serum can in some donors contain heterophilic antibodies which cross-links the capture and detection antibody. 

- The use of Tween in the PBS when washing the plates. Tween is never recommended if you use Mabtech pre-coated plates or coat yourself but use EtOH preactivation.

- If you activate your cells prior to adding them into the elispot wells, the cell supernatant can contain large quantities of the cytokine analyzed, resulting in a general darkening of the membrane. I know from experience with human cells that IFNg secretion begins just around 30min after the cells have been mixed with peptides. As a result, if you mix cells and stimuli, then prepare your elispot plate and finally add cells+stimuli 40 min later, the end result could high backgrounds. 

- Sometimes cell can be pre-activated in vivo. As a result, the splenocytes could have been actively producing IFNg at the time the cells were frozen. This continues even after thawing of the cells. During the 1h post thawing, IFNg will be released into the cell tubes and you can end up with rather large amounts of free IFNg in your cell solution. This obviously then darkens the membrane. One way to get rid of this problem is to wash the cells right before adding them into the ELISpot plates.

- Some peptides are known to cause dark membrane staining. So do you see this darkening only for some peptides? Or do you see it in all wells of the plate? We believe this could be related to high concentration of DMSO, but other factors could be in play as well. High DMSO can cause the membrane to leak and detection antibodies go deeper into the PVDF membrane. The result is strong membrane darkening. One should not use higher than 0.4% DMSO. In case your peptide needs high DMSO to be soluble, just dissolve the peptide in a small amount high concentration DMSO. Then dilute it down in PBS. The peptide will remain soluble even below 0.4%. What you also can do is lower concentration of the peptide down to 1ug/ml. That most often works just as well as 5ug/ml. 

 

So, questions: Are you using Tween? Possible you have invivo activated splenocytes? Is the darkening of the membrane happening all wells, or just in some peptides activated wells?  

 

best,

Christian

  

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Dear Christian, 

Thank you for your reply. I am not using Tween as Mabtech does not recommend to add tween. The darkening of the membrane was not happening in all wells. I have 8 replicate wells with the same samples and conditions. 5 wells showed high background and another 3 wells perfectly looked good. It happened to positive controls and samples. After thawing my splenocytes, I am waiting 1 hour or more in 37C. After that i washed the splenocytes and counted them. I firstly added stimuli then cells to the wells. I was not using peptide for my experiment. I was using purified antigen as stimuli. I am using Fetal Bovine serum with RPMI. For positive control, I was using PHA-M (10ug/ml). Actually the wells that did not contain any high background gave very good true spots. I was expecting like that. But why other wells have high background with same samples and conditions. After adding stimuli to the wells, I started washing the cells and counted. It may take 20-30 minutes. 

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ok, so some wells look really good and some have this darkening effect, including some positive control wells. 

Due to the fact that some look great, this sort of removes the possability of problems with the FBS, invivo-activated splenocytes or soluble cytokines in the cell supernatant.

Can you post any images? Please try to extract the high resolution images from the reader. Drag those into a PPT, write a comment how many cells and stimili below. I can then maybe identify the problem. Please include both problematic well, and well you say is perfect. 

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Dear Christian, 

Thank you for your reply. Actually I overcome the background problem. I used to incubate the splenocytes for 24 hours for stimulation. Last week, I tried with 18 hours of stimulation with the same conditions that i used before. Additionally, I used 10 minutes incubation with substrate and 1 minute incubation with PBS for every wash steps. Therefore, the background is gone. All the wells look very good and spots are so obvious. 

Thank you again for your response. 

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2 minutes ago, Shaikh Terkis Islam Pavel said:

Dear Christian, 

Thank you for your reply. Actually I overcome the background problem. I used to incubate the splenocytes for 24 hours for stimulation. Last week, I tried with 18 hours of stimulation with the same conditions that i used before. Additionally, I used 10 minutes incubation with substrate and 1 minute incubation with PBS for every wash steps. Therefore, the background is gone. All the wells look very good and spots are so obvious. 

Thank you again for your response. 

Excellent news! Come back anytime. The 10min incubation with substrate was probably very wise. 

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Guest dirty problems

Dear all,

I think I'm having the same problem as Christian.

I'm doing ELISPOT for detecting IFN-g secreting cells. The conditions are as follow: I use the ELISPOT product code 3321-4APT-2. In each well I add:

80 ul RPMI (+ Gln, 10% FBS, Pen Strep, Sodium Piruvate and 2-mercaptoethanol)

100 ul PPD antigen (diluted in PBS and PBS 0,01%FBS)

50000 cells

I incubate the cells ON at 37ºC and 5% CO2. 

I follow the isntructions regarding washes and incubation times. After adding the substrate, I see wells with a dark grey shadows on the bottom. I don't know how to get rid of them and what is the element is causing this issue.

I would appreciate some advice from you.

Sincerely,

Silvia Pérez

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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

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