Rosettesep technology

IFN-γ ELISPOT assays on Ugandan samples were performed as described previously.^{44 (link)} Briefly, CD8⁺ T cells and APC were negatively selected from PBMC by one of two methods. From 2004 to 2008, cryopreserved PBMC were thawed and then depleted of CD4⁺ and CD56⁺ cells by magnetic bead separation (Miltenyi Biotec, Auburn, CA, USA). We determined empirically a priori that CD56+ cell depletion reduced the background in the assay. From 2009 to 2012, fresh whole blood was depleted of CD4⁺ cells using RosetteSep technology from StemCell Technologies. Using RosetteSep, we determined empirically a priori that CD56+ depletion was not necessary to eliminate background in the assay. CD4-depleted PBMC (250,000 cells per well) were then used as the source of responding CD8⁺ T cells and APC in an IFN-γ ELISPOT assay, using overlapping synthetic peptide pools consisting of 15-mers overlapping by 11 aa (final concentration 5 μg/ml) as a source of antigen. The ELISPOT assay was performed once on each donor. All determinations were performed in duplicate. Positive responses were defined as those that are 2SD above the media control, and greater than 10 SFU, which is a similar but more conservative rule than we have utilized previously in cohorts of young Ugandan children.^{44 (link)}