Enrichment and Isolation of T and NK Cells

CD4 positive and CD8 positive T cells were negatively enriched from fresh leukopacks (American Red Cross, Columbus, OH) by 30-minute incubation with Rosette Sep CD4 T (or CD 8 T) cell enrichment cocktail (Stem Cell Technologies, Vancouver, BC), followed by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. CD4⁺ (or CD8⁺) cells were then isolated to purity by positive selection using EasySep Human CD4 (or CD 8) Positive Selection Kit (Miltenyi Biotec, Auburn, CA). Purity of isolated CD4⁺ and CD8⁺ cells were evaluated by flow cytometry using CD3 V450 and CD4 APC or CD8 APC, and purity was validated to be greater than 95 percent. NK cells were negatively enriched from fresh peripheral blood leukopacks (American Red Cross: Columbus, OH) by 30-minute incubation with RosetteSep cocktail (Stem Cell Technologies: Vancouver, BC) followed by density gradient centrifugation as previously described (18 (link)), and used immediately. Purity of the isolated NK cells was evaluated using CD56 APC and CD3 V450, and validated to be greater than 81 percent.

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del Campo S.E., Levine K.M., Mundy-Bosse B.L., Grignol V.P., Fairchild E.T., Campbell A.R., Trikha P., Mace T.A., Paul B.K., Jaime-Ramirez A.C., Markowitz J., Kondadasula S.V., Guenterberg K.D., McClory S., Karpa V.I., Pan X., Olencki T.E., Monk J.P., Mortazavi A., Tridandapani S., Lesinski G.B., Byrd J.C., Caligiuri M.A., Shah M.H, & Carson WE I.I.I. (2015). The Raf Kinase Inhibitor Sorafenib Inhibits JAK-STAT Signal Transduction in Human Immune Cells. Journal of immunology (Baltimore, Md. : 1950), 195(5), 1995-2005.

Publication 2015

Ficoll-Paque

Blood Cd8 cells Density gradient centrifugation Ficoll Flow cytometry Human Nk cells Stem cell T cell

Corresponding Organization : The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute

Other organizations : Nationwide Children's Hospital, Neurological Surgery, The Barbara Ann Karmanos Cancer Institute, Barnes-Jewish Hospital, University of Hawaii System

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Variable analysis

independent variables

Incubation with Rosette Sep CD4 T (or CD 8 T) cell enrichment cocktail
Ficoll-Paque density gradient centrifugation
Positive selection using EasySep Human CD4 (or CD 8) Positive Selection Kit

dependent variables

Purity of isolated CD4+ and CD8+ cells
Purity of isolated NK cells

control variables

Fresh leukopacks (American Red Cross, Columbus, OH) for CD4+ and CD8+ T cells
Fresh peripheral blood leukopacks (American Red Cross: Columbus, OH) for NK cells

positive controls

None specified

negative controls

None specified

Annotations

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