It may depends on the frequency of antigen-specific cells. Sometimes, I had to expand the antigen-specific cells on culture with peptides and IL-2 for almost 2 weeks before testing them on the ELISPOT plates. In that case, they were all frozen-thawed cells.

Maybe try to look at the following manuscript – they extensively analyze what is the optimal thawing procedure for downstream Elispot:

Ramachandran H1, Laux J2, Moldovan I3, Caspell R4, Lehmann PV5, Subbramanian RA6. Optimal thawing of cryopreserved peripheral blood mononuclear cells for use in high-throughput human immune monitoring studies. Cells. 2012 Jul 25;1(3):313-24. doi: 10.3390/cells1030313.

Are you resting the responder cells before you put them in the ELISpot assay?  I find you get much better results if you rest the cells over night and then do you live cell count, adjust the concentration and use them in the elispot assay.  

Hi Josh

Definitely agree with Rebecca, I have used freeze/thawed PBMCs for peptide stimulations of IFNy ELISpot assays by resting them in complete media overnight in the 37C incubator and never had any problems.

If that doesn't work, you may have a problem with the costimulation from APCs.. I found using anti-human CD28/CD49d in the culture helped detecting IFNy production on flow cytometry, but never needed to try it for ELISpots.

Good luck!

Hi Josh; Great previous advice from Rebecca and Aleks. Overnight resting can recover functionality of previously frozen PBMC via multiple mechanisms. A paper by Santos et al. gives a good protocol and shows what this can do: Cells. 2014 Dec 24;4(1):1-18. doi: 10.3390/cells4010001. Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. 

FCS can also be responsible for suppressed responses. Is the FCS you use in the freezing medium the same as in your assay (same batch etc.)? Did you ever pretest it? If not, you may want to consider serum-free freezing medium (see here: Filbert et al; Cancer Immunol Immunother. 2013 Apr;62(4):615-27. doi: 10.1007/s00262-012-1359-5. 

Serum-free freezing media support high cell quality and excellent ELISPOT assay performance across a wide variety of different assay protocols.

Sylvia

In our experience resting can be sometimes detrimental to the ELISPOT assay results and this mainly depends on what antigen is used and the PBMC source. Resting however performs  Ok with healthy individuals PBMC using classical short CD8 peptides.  You will need to check with your specific antigen and PBMC source if it is beneficial or not in your assay system.

We recommend using serum free freezing processes as better ELISPOT assay results are observed. FCS can be sometimes toxic to cells eliminating antigen specific responses. Please see reference

Zhang W, Caspell R, Karulin AY, Ahmad M, Haicheur N, Abdelsalam A, Johannesen K, Vignard V, Dudzik P, Georgakopoulou K, Mihaylova A, Silina

K, Aptsiauri N, Adams V, Lehmann PV, McArdle S. ELISPOT assays provide reproducible results among different laboratories for T-cell immune

monitoring – even in hands of ELISPOT-inexperienced investigators. J. Immunotoxicology. 2009. 6:227-34

Magdalena

Josh, if you get good responses with your PHA control, you can conclude that you have both antigen-presenting cells and responding cells in your assay. For PHA to work, you need functional APCs. The problem seems to be on the stimuli used. If your stimulus is specific antigen, it may be that your antigen-specific T cells are depleted or over-stimulated.

Hi Josh;  I agree completely with the advice from Rebecca and Aleks. resting cells for few hours can recover PBMC's from their shocking with the freezing as Sylvia mentioned above by multiple mechanisms.  The second tip is to count viability after resting and try to get rid of dead cells. And I agree with Maria, optimize the No. and time of your T cells and try also to use different responders cells, I mean different immunocompetent sources.

Dear Josh,

    I suggest that the responder cells remain in the culture for an overnight at 37 ºC in the complete medium. Thereafter, stimulate your responder cells with appropriate stimulator and expand the antigen-specific cells in culture before you perform cell plating in the ELISPOT wells. In this regard, optimization of stimulator concentration and time of stimulation are required. Next, apply higher cell density of live stimulated cells in the medium containing reduced FCS (5%) without antibiotics, and culture them in the ELISPOT wells for one day extra at 37 ºC and 5% CO2.

I agree that you should try to  thaw the cells and rest them in medium over night. Then count them and do the stimulation/assay. This should work as long as the freezing and thawing conditions are OK. I suggest you use ConA or activating anti-CD3 as these require B-7 costimulation on APCs, PHA does not as it also stimulates purified T cells. This will give you an indication whether your costimulatory conditions are OK. Even better may be to use a peptide pool designed to stimulate T cells from donors with a variety of HLA types.

Thank you all very much for the wonderful responses!  I have pulled many of the publications you suggested and will research what would be best for our causes.

As we are typically using this method for yIFN, I think I will try to 'rest' the cells overnight.  Thank you all for that wonderful suggestion!

Sylvia: yes the FCS we use to freeze is the same as the FCS we use in our assay media.  We do use Premium Select Grade from Atlanta Biologics.  We do not QC it, but from what I understand the company goes through a rigorous QC process and lot matches our new orders.  Can you recomend any serum free freezing media?

Magdelena:  As we work on a transplant model, we tyipically use allogeneic PBMC as a source of stimulation.  I am not sure I fully understand your advice about resting.   Although we typically test for IFNy, I would like to extend our cytokine measurements; would resting not work for measuring other cytokines?

I am very excited to have so much support from the community.  Thank you again all!!

Josh

Hi Josh; The Filbert publication listed above contains various options for serum-free freezing medium, including a BSA-based prepare-it-yourself option. 

Re Overnight resting, there are excellent publications out including one by Romer in Blood from 2011, Vol 118, demonstrating multiple beneficial effects of resting, here in a CD4 setting. Key is a correct protocol and understanding of the mechanisms ongoing on the cellular level, and that cells that are undergoing apoptosis will die. Another excellent publication demonstrating the effects of resting via flow and for various cytokines is by Kutscher in PLOS One 2 years ago. 

Good luck for your work! Sylvia