Protocol detail

Multiparameter Flow Cytometry Assay

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Conjugated antibodies and staining reagents included MIP-1β-PE, CD3-PB, CD3-PE-Cy7, CD3-PerCP-Cy5.5, CD3-APC-Cy7, CD3-Horizon V450, CD4-PerCP-Cy5.5, CD4-AmCyan, CD4-FITC, CD8α-APC, CD8α-APC-Cy7, CD8α-AlexaFluor700, CD8α-FITC, CD8α-APC-H7, CD69- ECD (Beckman Coulter), CD20-Horizon V450, CCR7-FITC (R&D Systems), CD95-APC, CCR7-PerCP-Cy5.5, CD28-PE-Cy7 (eBioscience), granzyme B-AlexaFluor700, perforin-FITC (MabTech), IFNγ-PE-Cy7, TNFα- AlexaFluor700, IL-2-APC, CD95-PE, CD95-APC, and Aqua LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). All reagents are from BD Biosciences unless indicated otherwise. For construction of monomers and tetramers, the following peptides were synthesized and purified to >95% by HPLC by New England Peptide LLC: p11C (CTPYDINQM), p54AS (TVPWPNASL), p54E660 (TVPWPNETL), p68A (STPPLVRLV), and TL8 (TTPESANL). The monomers and tetramers were prepared as previously described [85] (link), [86] (link). Tetramers were prepared using either streptavidin-PE (Prozyme), -APC (Prozyme), -AlexaFluor488 (Invitrogen), or -Qdot655 (Invitrogen). Monomers used in surface plasmon resonance studies were further quantified using an RC DC protein kit (Bio-Rad).

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Osuna C.E., Gonzalez A.M., Chang H.H., Hung A.S., Ehlinger E., Anasti K., Alam S.M, & Letvin N.L. (2014). TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01 + Rhesus Monkeys. PLoS Pathogens, 10(4), e1004069.

Publication 2014

MIP-1β-PE CD3-PE-Cy7 CD3-PerCP-Cy5.5 CD4-PerCP-Cy5.5 CD4-FITC CD69- ECD CCR7-FITC CD95-APC CCR7-PerCP-Cy5.5 CD28-PE-Cy7 perforin-FITC Aqua LIVE/DEAD Fixable Dead Cell Stain -AlexaFluor488 -Qdot655

Antibodies Cell Cy5 5 Fitc Granzyme b Hplc Ifnγ Peptide Perforin Protein Stain Streptavidin Surface plasmon resonance Tetramers Tnfα

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Variable analysis

independent variables

Conjugated antibodies and staining reagents
Peptides used for construction of monomers and tetramers

dependent variables

Measured outcomes (not explicitly mentioned)

control variables

Peptides synthesized and purified to >95% by HPLC
Monomers and tetramers prepared as previously described
Monomers further quantified using an RC DC protein kit

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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