Cd107a pe cy5

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The CD107a PE-Cy5 is a fluorescently-labeled monoclonal antibody that can be used to detect the cell surface expression of the CD107a (LAMP-1) protein. CD107a is a marker of cellular degranulation and is commonly used to assess the functional activity of cytotoxic T cells and natural killer cells.

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3 protocols using cd107a pe cy5

Multiparametric Immune Cell Phenotyping

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Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).

Demers K.R., Makedonas G., Buggert M., Eller M.A., Ratcliffe S.J., Goonetilleke N., Li C.K., Eller L.A., Rono K., Maganga L., Nitayaphan S., Kibuuka H., Routy J.P., Slifka M.K., Haynes B.F., McMichael A.J., Bernard N.F., Robb M.L, & Betts M.R. (2016). Temporal Dynamics of CD8+ T Cell Effector Responses during Primary HIV Infection. PLoS Pathogens, 12(8), e1005805.

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Characterization of T cell kinetics post-viral vector

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For the characterisation of T cell kinetics after IV viral vector, freshly isolated PBMC were stimulated with anti-CD28 and anti-CD49d at 1μg/mL (Becton Dickinson), 200μg/mL of CD107a-PeCy5 (eBioscience) together with a pool of all 56 peptides of the T9/96 strain P. falciparum TRAP antigen (final concentration 5µg/mL) for 16-20 hours (32 (link)), 5μg/ml Staphylococcal enterotoxin B (SEB) (Sigma Aldrich) or media (unstimulated). Brefeldin A and monensin (eBioscience), both at 1 μg/mL, were added after two hours. For lymphocytes used in matched PBMC and FNA characterisation, no peptide stimulation was performed.
Following surface staining, fixation and intracellular staining (see Supplementary Materials and Methods for antibody list), acquisition was performed using an LSRII or LSRFortessa X-20 SORP (BD Biosciences). At least 500,000 events were acquired per sample, with data analysed on FlowJo version 10 (BD Biosciences). Lymphocytes were gated on live, size, and singlet (FSC-A vs FSC-H and FSC-A vs FSC-W) and dead cells (Live/Dead amine reactive+), monocytes (CD14+) and B cells (CD19+) were excluded. Cells were subsequently gated on CD45+ CD3+ CD8+ T cells.

Noé A., Datoo M.S., Flaxman A., Husainy M.A., Jenkin D., Bellamy D., Makinson R.A., Morter R., Ramos Lopez F., Sheridan J., Voukantsis D., Prasad N., Hill A.V., Ewer K.J, & Spencer A.J. (2022). Deep Immune Phenotyping and Single-Cell Transcriptomics Allow Identification of Circulating TRM-Like Cells Which Correlate With Liver-Stage Immunity and Vaccine-Induced Protection From Malaria. Frontiers in Immunology, 13, 795463.

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Multiparameter Flow Cytometry for NK Cells

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For surface and intracellular staining of NK cells the following mouse anti-human fluorescent-labeled Abs were used: CD2-PE-Cy7 (clone TS1/8), CD56-BrilliantViolet421 (clone HCD56), CD56-AF488 (clone 5.1H11), CD56-PE-Cy7 (clone 5.1H11), CD57-PE (clone TB01), HLA-DR-FITC (clone LN3), HLA-DR-PE (clone L243), IFN-gamma-FITC (clone 4S.B3), KI-67-PE (clone Ki-67), NKp46-FITC (clone 9E2) (Sony Biotechnology, USA), CD16-PE (Sorbent, Russia), CD16-APC (clone REA423), CD56-APC (clone REA196), CD57-FITC (clone TB03), CD57-APC (clone TB03), CD57-PE-Vio770 (clone TB03) (Miltenyi Biotec, Germany), CD44-F (clone J.173, Immunotech, USA), CD56-PE (clone C5.9, Dako, USA), CD107a-PE-Cy5 (clone H4A3), NKp30-PE (clone P30-15) (eBioscience, USA), CD161-AlexaFluor647 (clone HP-3G10), Granzyme B-Alexa Fluor 647 (clone GB11), KIR2DL2/DL3-PE (clone DX27) (Biolegend, USA), FcεRIγ-FITC (polyclonal, Milli-Mark, Merck, Germany), NKG2A-PE (clone 131411), NKG2C-AlexaFluor 488 (clone 108724), NKG2C-PE (clone 134591) (R&D Systems, USA). Supernatant of hybridoma producing Abs to human KIR2DL1 was kindly provided by prof. Miguel Lopez-Botet (Universitat Pompeu Fabra, Barcelona, Spain). Sheep anti-mouse IgG-FITC and PE (Sigma-Aldrich, USA) were used as second Abs for anti-KIR2DL1. IgG1-PE (clone IS5-21F5, Miltenyi Biotec, Germany) was used as isotype control for NKG2C-PE Ab staining.

Kobyzeva P.A., Streltsova M.A., Erokhina S.A., Kanevskiy L.M., Telford W.G., Sapozhnikov A.M, & Kovalenko E.I. (2020). CD56dimCD57−NKG2C+ NK cells retaining proliferative potential are possible precursors of CD57+NKG2C+ memory-like NK cells. Journal of leukocyte biology, 108(4), 1379-1395.

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