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Cd107a apc

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CD107a-APC is a fluorescently labeled antibody that binds to the CD107a (also known as LAMP-1) surface antigen. CD107a is a marker of lysosomal-associated membrane protein 1 and is expressed on the cell surface during degranulation of cytotoxic T cells and natural killer cells.

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Lab products found in correlation

Ifnγ pe, by Miltenyi Biotec (3 mentions) Hla dr percp, by Miltenyi Biotec (2 mentions) E cadherin apc, by Thermo Fisher Scientific (2 mentions) N cadherin pe, by Abcam (2 mentions) Monensin, by Thermo Fisher Scientific (2 mentions) Fixable aqua dead stain, by Thermo Fisher Scientific (2 mentions) Inside stain kit, by Miltenyi Biotec (2 mentions) Cd4 percp vio700, by Miltenyi Biotec (1 mentions) 7 aminoactinomycin d 7 add, by BD (1 mentions) Cd3 fitc, by Miltenyi Biotec (1 mentions) Cd11b allophycocyanin apc vio770, by Miltenyi Biotec (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions) Cytexpert software, by Beckman Coulter (1 mentions) Fitc conjugated annexin 5, by BD (1 mentions) Cd8a apc, by Miltenyi Biotec (1 mentions) Cd105 pe, by Miltenyi Biotec (1 mentions) Cd80 apc, by Miltenyi Biotec (1 mentions) Epcam vioblue, by Miltenyi Biotec (1 mentions) Ccr7 fitc, by Miltenyi Biotec (1 mentions) Dnam 1 pe, by BioLegend (1 mentions)

8 protocols using cd107a apc

1

Flow Cytometric Analysis of Immune Cells

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The following Abs against mouse molecules were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany): CD3-FITC, NK1.1-PerCP-Vio700, CD11b-allophycocyanin (APC)-Vio770, CD27-PE-Vio770, CD107a-APC, IFN-γ-PE, CD4-PerCP-Vio700, and CD8a-APC. 7-Amino-actinomycin D (7-ADD) and FITC-conjugated annexin V were purchased from BD Biosciences (San Jose, CA, USA). Flow cytometric analysis was performed using a Cytoflex flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and data analysis was performed with CytExpert software (Beckman Coulter, Inc.).
Jang S.H., Oh M.S., Baek H.I., Ha K.C., Lee J.Y, & Jang Y.S. (2018). Oral Administration of Silk Peptide Enhances the Maturation and Cytolytic Activity of Natural Killer Cells. Immune Network, 18(5), e37.
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2

Multi-Marker Cytometry Profiling

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For cytofluorimetric analysis, cells were stained with surface antibodies in PBS 5% FCS for 20 min at 4 °C. The following antibodies were used: CD133-APC, EpCAM -VioBlue, CD105-PE, CD90-VioBlue, IFNγ-PE, CD107a-APC, CCR7-FITC, CD163-PerCP-Vio700, CD206-VioBlue, HLA-DR-PerCP, CD80-APC (MIltenyi Biotec); CD56-PC7, NKp30-PE (Beckman Coulter); CD29-PE (ImmunoTools); CD146-PC7, CD73-FITC, NKp46-V450 (BD Biosciences); E-cadherin-APC (Thermo Fisher Scientific); N-cadherin-PE (Abcam); DNAM1-PE (Biolegend).
Fiore P.F., Vacca P., Tumino N., Besi F., Pelosi A., Munari E., Marconi M., Caruana I., Pistoia V., Moretta L, & Azzarone B. (2021). Wilms’ Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages. Cancers, 13(2), 224.
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3

Comprehensive Immune Profiling by Cytofluorimetry

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For cytofluorimetric analysis, cells were stained with surface antibodies in PBS containing 5% FCS for 20 min at 4 °C. The following antibodies were used: IFNγ-PE, CD107a-APC, HLA-DR-PerCP, CD69-FITC, PD-1-APC (Miltenyi Biotec); CD56-PC7, NKp30-PE (Beckman Coulter); NKp46-V450, CD16 PerCP-Cy5,5, CD155-AF647, PD-L1 PE-CF59 (BD Biosciences); E-cadherin-APC (Thermo Fisher Scientific); N-cadherin-PE (Abcam); NKG2D-PE/Dazzle, DNAM1-PE, CD112-PerCP-Cy5,5; MICA/B PC7 (Biolegend); ULBP3 AF405, ULBP2/5/6 PE (R&D).
Fiore P.F., Di Pace A.L., Conti L.A., Tumino N., Besi F., Scaglione S., Munari E., Moretta L, & Vacca P. (2022). Different effects of NK cells and NK-derived soluble factors on cell lines derived from primary or metastatic pancreatic cancers. Cancer Immunology, Immunotherapy, 72(6), 1417-1428.
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4

Multicolor Immunofluorescence Staining of NK Cells

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CD56+ enriched cells resuspended in 1 mL of PBS were added on Poly-L-Lysine (PLL) treated coverslips. After incubation, the attached cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT). After extensively washing with PBS, cells were incubated with Blocking Solution (1 mg/ml BSA, 3% goat serum, 0.1%Triton X-100 in PBS) for 30 min at RT. Next, the cells were incubated with antibodies in Blocking Solution for 1 hr at 4°C and washed three times with PBS. Immunostainings were performed using the following fluorophore-conjugated antibodies: CD3-VioGreen (clone REA613), CD107a-APC (clone REA792), and PD-1-VioBright FITC (clone PD1.3.1.3) all from Miltenyi, and PD-1-Alexa Fluor 700 (clone EH12.2H7, BioLegend), granzyme B-PE (clone REA226, Miltenyi) and Hoechst (Molecular Probes). Preparations were mounted with Fluoromount-G (EMS), and confocal fluorescence images were acquired using a Zeiss LSM 880 Spectral Confocal Microscope. NK cells and NKT cells were differentiated by the absence/presence of CD3 labeling.
Pesini C., Hidalgo S., Arias M.A., Santiago L., Calvo C., Ocariz-Díez M., Isla D., Lanuza P.M., Agustín M.J., Galvez E.M., Ramírez-Labrada A, & Pardo J. (2022). PD-1 is expressed in cytotoxic granules of NK cells and rapidly mobilized to the cell membrane following recognition of tumor cells. Oncoimmunology, 11(1), 2096359.
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5

Flow Cytometry Analysis of Surface Markers

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Flow cytometry was used for the analysis of surface marker or transgene expression. The following antibodies were used: CD3-APC (OKT3, BioLegend, San Diego, CA, USA), CD16-PE (REA423, Miltenyi), CD56-APC (AF12-7H3, BioLegend; HCD56, Miltenyi Biotec), CD56-FITC (REA196 Miltenyi), CD56-PE (B159, Becton Dickinson, BD, Heidelberg, Germany), Ganglidiomab-PE (Gangliomab-phycoerythrin (PE) monoclonal antibody was kindly provided by H. Lode and N. Siebert (46 (link)) and PE-conjugated), IgG, F(ab′)2 (Biotin-SP-conjugated, Jackson ImmunoResearch, West Grove, PA, USA), Ganglioside GD2-APC (14G2a, BioLegend), Ganglioside GD2-PE (14G2a, BioLegend), and Annexin V-PE (BD Biosciences, Franklin Lakes, USA) and the secondary antibodies Streptavidin-APC (BioLegend) and Streptavidin-PE (eBioscience, San Diego, CA, USA). For staining of the degranulation marker CD107a, cocultures were incubated with CD107a-APC (Miltenyi) for 1 h at 37°C and 5% CO2 with shaking. Afterward, Brefeldin A (1:1,000; BioLegend) and Monensin (1:1,000; BioLegend) were added for an additional incubation for 4 h at 37°C and 5% CO2 with shaking. Stained cells were measured via FACSCalibur cytometer (Becton Dickinson, BD) or CytoFLEX (Beckman Coulter) and analyzed via FlowJo software (Tree Star Inc., Ashland, OR, USA).
Rudek L.S., Zimmermann K., Galla M., Meyer J., Kuehle J., Stamopoulou A., Brand D., Sandalcioglu I.E., Neyazi B., Moritz T., Rossig C., Altvater B., Falk C.S., Abken H., Morgan M.A, & Schambach A. (2021). Generation of an NFκB-Driven Alpharetroviral “All-in-One” Vector Construct as a Potent Tool for CAR NK Cell Therapy. Frontiers in Immunology, 12, 751138.
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6

Cytokine and Degranulation Analysis

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Cytokine production and degranulation were analyzed by means of flow cytometry. NK cells (2 × 104) per well were seeded in 96-well round-bottom plates alone, as a control, or together with 1 × 104 K562 cells and cultivated for 4 h in RPMI1640 supplemented with Monensin (eBioscience) and CD107a-APC (Miltenyi) according to the user manuals. After labeling with Fixable Aqua Dead Stain (Life Technologies) to exclude dead cells during the analysis, the cells were fixed, permeabilized and stained for IFN-γ, TNF-α and CD56 (Inside Stain Kit, Miltenyi). The latter marker was used to discriminate NK cells from co-cultivated K562 target cells.
GRANZIN M., SOLTENBORN S., MÜLLER S., KOLLET J., BERG M., CERWENKA A., CHILDS R.W, & HUPPERT V. (2015). Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy. Cytotherapy, 17(5), 621-632.
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7

Multiparametric NK Cell Immunophenotyping

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The following mouse anti-human fluorescent-labeled antibodies were used for surface cell staining: CD56-APC-Vio770 (clone REA196), CD57-VioBlue (clone TB03), CD107a-APC (clone REA792), HLA-DR-PE-Vio770 (clone REA805), IFNγ-PE (clone 45-15), KIR2DL1-APC (clone REA284), KIR2DL2/DL3-PE (clone DX27), NKG2C-VioBright (clone REA205), HLA-E-PE (clone REA1031) (Miltenyi Biotec, Bergisch Gladbach, Germany); CD56-BrilliantViolet 421 (clone 5.1 H11), CD57-PE (clone HNK-1), CD57-APC (clone HNK-1) (Sony Biotechnology, San Jose, CA, USA); NKG2C-PE (clone 134591) (R&D Systems, Minneapolis, MN, USA).
PBMC/NK cells were washed in the PBA staining buffer (PBS containing 0.5% BSA (bovine serum albumin) (PanEco, Moscow, Russia) and 0.01% sodium azide (PanEco, Moscow, Russia) and incubated with antibodies for 30 min on ice in a PBA then washed twice in the same buffer before measurement. Flow cytometry analysis was carried out on a MACSQuant 10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) equipped with 405 nm, 488 nm, and 635 nm lasers.
Alekseeva N.A., Streltsova M.A., Vavilova J.D., Ustiuzhanina M.O., Palamarchuk A.I., Boyko A.A., Timofeev N.D., Popodko A.I, & Kovalenko E.I. (2024). Obtaining Gene-Modified HLA-E-Expressing Feeder Cells for Stimulation of Natural Killer Cells. Pharmaceutics, 16(1), 133.
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8

Cytokine and Degranulation Analysis

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Cytokine production and degranulation were analyzed by means of flow cytometry. NK cells (2 × 104) per well were seeded in 96-well round-bottom plates alone, as a control, or together with 1 × 104 K562 cells and cultivated for 4 h in RPMI1640 supplemented with Monensin (eBioscience) and CD107a-APC (Miltenyi) according to the user manuals. After labeling with Fixable Aqua Dead Stain (Life Technologies) to exclude dead cells during the analysis, the cells were fixed, permeabilized and stained for IFN-γ, TNF-α and CD56 (Inside Stain Kit, Miltenyi). The latter marker was used to discriminate NK cells from co-cultivated K562 target cells.
GRANZIN M., SOLTENBORN S., MÜLLER S., KOLLET J., BERG M., CERWENKA A., CHILDS R.W, & HUPPERT V. (2015). Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy. Cytotherapy, 17(5), 621-632.
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