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Guest Alok Tembhurne

Stimuli for ELISPOT

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Guest Alok Tembhurne

I want to do ELISPOT for IFN gamma  to assess T cell memory response against HPV 16 E6 peptides. I have a doubt about stimulating cells during ELISPOT. 

Monocytes are cells which function in presenting antigen to T cells and they will be present in PBMC population. We expect monocytes to take up the antigenic peptide and present it to T cells in context of respective MHC I/II molecule. However, some papers recommend isolation T cell clones  infected with Vaccinica virus expressing HPV 16 E6 protein. 

Do you have any suggestions for this? Also can you cite a paper where Mabtech ELISPOT kit has been used which may help in this.

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Peptide stimulation typically works very well in ELISpot. This is what most vaccine papers using this method employ. Given that the peptide induces a response it is very reliable. In every quality control at Mabtech we include the CEF peptide pool. As long as the donor is a responding one, it always works. One should make sure that the cells are of good quality. Viability should be over 84% and this includes both dead and apoptotic cells. 

By contrast, some researchers are investigating whole proteins. These need to go through much more extensive processing and presentation and here there can be limitations in getting the T-cells to respond in certain scenarios. One might need to go up very high in cell numbers/well and one might need to supplement with anti-CD28 antibody stimulation to see a clear response. Some go to even greater extent and load autologoous dendritic cells with antigen and add those in great numbers to isolated T-cells. Or in the case you are refering to: mix in cells infected with virus expressing the antigen. 

All of this is ofcourse possible but one should always start with the easy approach: Take your peptide and run an ELISpot at 250,000 cells/well and 400,000 cells/well. Titrate the peptide so you know you get optimum stimulatino. Usually stimulation is effective at 2ug/ml but go up to 4-5 just to be sure. Make sure your cells are in good condition. Get a precoated Mabtech IFNg ELISpot kit. Select an ALP kit like this one:

https://www.mabtech.com/products/human-ifn-gamma-elispot-pro-kit-alp_3420-2apt-2

For your initial testing don't coat yourself. Most often when ELISpot goes wrong it is when you make mistake in coating. Precoated kits from Mabtech have been done under optimal conditions which include the etoh activation. Performance is much much better compared to coating yourself but not doing the etoh. 

 

 

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Guest Alok Tembhurne

Dear Christian,

Thank you for your valuable suggestions. They will be helpful when I will start my assay. I understand that cell quality, number of cells and quantity of antigenic stimuli I quite important for expecting spots.

Now I understand why there is so much variation among research papers publishing their data and protocol.

Also, it is really kind of you suggesting which ELISPOT kit I should buy for my assay.

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Very good! We are here to help!

When you buy the kit we include a positive control that is anti-human CD3. A very reliable polyclonal activator of T-cells. Recommended cell number for this is 50,000-100,000 cells/well when analyzing human IFNgamma. So a littlebit less compared to antigen specific wells that typically need 250,000-400,000 cells/well. 

It is good to always include a few wells with a postivie control since it will tell you that the cells are in working order. 

Good luck!

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Guest Alok Tembhurne

Dear Christian,

My peptides have arrived and my Mabtech ELISPOT kit will arrive shortly. I have some technical doubts.

1. During storage of PBMC population, do I need to add recombinant human Interleukin - 2 ? If yes, should I use 5U/ml or 20U/ml along with 100 U/ml penicillin and 100mg/ml streptomycin?

2. While setting up my ELISPOT assay, should I use 5% pooled Human serum or 10 % FCS in RPMI 1640 ?

3. Do I need to use anti CD-28 additionally during EISPOT assay?

Thanking you in advance.

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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

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Guest Alok Tembhurne

Well said!

Really good suggestions before starting off with my experiment. One more thing I would like to ask:

Should I use cryopreserved PBMCs or freshly isolated PBMCs.

Its bewildering hold up, as my neighbouring labs in the institute tell me to use freshly isolated ones, which might increase complexity as how many samples I get in a day and freeze thawing of reagents and materials (which may affect their efficiency). Please suggest on this.

Sorry to bother you again!

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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!

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Guest Alok Tembhurne

Hey,

Thanks a lot.

It is really nice hearing from you on this. If I have any other doubts, I'll write you. 

Regards,

Alok

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