Hi , My over all experimental goal is to define Zika virus-specific T cell responses in macaques infected with Zika virus. We also have control macaques that were not infected with Zika. We used PBMCs obtained from 14-28 days after Zika infection. PBMCs and stimulants were arranged in the Elispot plate as shown below.
Viability - determined with trypan blue assay
PBMC preservation - frozen in DMEM with 10% FBS, antibiotic, L-glutamine+10% DMSO
Elispot assay: stimulant and PBMCs mixed together prior to adding to well, incubated ~17 hours overnight>
Expected results: should see lots of spots in all CD3 wells, and spots in all the wells from a Zika animal stimulation with UV-inactivated Zika virus or Zika peptides
Actual results: only see spots in PBMCs from 1 Control animals stimulated with CD3.
No spots in Zika PBMCs stimulated with CD3 Ab. No spots with Zika stimulants.
Any suggestions?
Do you think changing the format to Cultured Elispot help?
Thanks for your response.
Saswati
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