Anti nkp30 apc

Manufactured by BioLegend

Sourced in United States

Anti-NKp30-APC is a fluorochrome-conjugated antibody that binds to the NKp30 receptor on natural killer (NK) cells. NKp30 is a key activating receptor involved in the recognition and killing of tumor cells and virus-infected cells by NK cells.

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4 protocols using anti nkp30 apc

Comprehensive Immunophenotyping of haNK Cells

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The cetuximab (Bristol-Myers Squibb, New York, NY), trastuzumab (Genentech, San Francisco, CA), pertuzumab (Genentech) and isotype (rituximab, Genentech) antibodies were purchased from the National Institutes of Health Pharmacy. Flow cytometry of haNK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Flow cytometry of tumor cells was performed on a BD FACSVerse flow cytometer (BD Biosciences) and analyzed in BD FACSuite (BD Biosciences). Staining of haNK cells was performed with four panels, and the antibodies used were: Anti-CD56-PErCP-Cy5.5, anti-CD16-APC-Cy7, anti-NKG2D-APC, anti-NKG2D-FITC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-CD158a-PE, anti-CD158d-PE, anti-CD158j-APC, anti-PD-1-PE-Cy7, anti-PD-L1-APC, and anti-MICA-PE; all were obtained from BioLegend (San Diego, CA). Anti-CD56-PE, anti-CD16-PE-Cy7, CD11a-PE, Ki67-FITC, anti-HLA-ABC-FITC and anti-4-1BB-BV421 were obtained from BD Biosciences. In controlled experiments to detect CD16 on haNK cells, the anti-CD16 MAb was CD16-FITC (BD Biosciences) and the isotype control antibody was MIgG1k-FITC (BD Biosciences).

Jochems C., Hodge J.W., Fantini M., Fujii R., Maurice YM I.I., Greiner J.W., Padget M.R., Tritsch S.R., Tsang K.Y., Campbell K.S., Klingemann H., Boissel L., Rabizadeh S., Soon-Shiong P, & Schlom J. (2016). An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele. Oncotarget, 7(52), 86359-86373.

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Multiparameter Flow Cytometry of NK Cells

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Flow cytometry of NK cells was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed in FlowJo 9.9 (TreeStar Inc., Ashland, OR). Staining of NK cells was performed with four panels, and the antibodies used were: anti-CD56-BV605, anti-NKG2D-APC, anti-CD107a-PE-Cy7, anti-GranzymeB-FITC, anti-perforin-APC, anti-NKp46-PE-Cy7, anti-NKp30-APC, anti-NKp44-PE, anti-2B4-FITC, anti-4-1BB-PerCP-Cy5.5, anti-CD95-BV421, anti-Tim3-BV421, anti-TRAIL-PE, anti-CD122-PerCP-Cy5.5, anti-CD122-BV510, anti-CD95L-PE, anti-Ki67-BV421, anti-CD40L-BV510, anti-PD-1-PE-Cy7; all were obtained from BioLegend (San Diego, CA). Anti-CD16-APC-H7, PD-L1-PE-Cy7, and CD11a-FITC, were obtained from BD Biosciences (San Jose, CA). Anti-NKG2A-PE was from R&D Systems (Minneapolis, MN), and anti-CD158a-PerCP-Cy5.5 from eBioscience.

Jochems C., Tritsch S.R., Pellom S.T., Su Z., Soon-Shiong P., Wong H.C., Gulley J.L, & Schlom J. (2017). Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824). Oncotarget, 8(43), 75217-75231.

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Multi-panel Flow Cytometry Analysis of PBMC

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25 ml of blood was collected prior to pancreaticoduodenectomy surgery and peripheral blood mononuclear cell (PBMC) were collected by density centrifugation. After defrosting, PBMCs were washed and re-suspended at 106cells/100 μl before staining with one of the following antibody panels. Panel 1; anti-DNAM-1 FITC (11A8 Biolegend) (5 µl); anti-PD-1 PE (EH12.2 H Biolegend) (3ul); anti-CD14/CD19 PE dazzle (HCD14/H1B19 Biolegend) (1.5 µl/2.5 µl); anti-NKp46 PerCP/Cy5.5 (Biolegend) (5 µl); anti-CD56 PEcy7(HCD56 Biolegend) (3 µl); anti-NKG2D APC (BD Biosciences) (5 µl); anti-CD3 AF700 (HIT3a Biolegend) (2.5 µl) and anti-CD16 Pacific Blue (3G8 Biolegend) (5 µl). Panel 2; anti-NKG2C FITC(11A8Milteny Biotec) (2µl); anti-CD94 PE (DX22 Biolegend)(1 µl); anti-CD14/CD19 PE-dazzle (HCD14/H1B19Biolegend) (1.5 µl/2.5 µl); anti-KIRS PerCP/Cy5.5 (HP-MA4 Biolegend)(5 µl); anti-CD56PECy7 (HCD56 Biolegend) (3 µl); anti-NKp30 APC (Biolegend)(5 µl); anti-CD3 AF700 (HIT3a Biolegend) (2.5 µl) and anti-CD57 Pacific Blue (HNK-1Biolegend) (3 µl).
Antibody stained samples were run on a BC Gallios flow cytometer within 1 h of staining. At least 100,000 events were acquired per sample using CytoSoftware, and analyzed offline using Kaluza v1.5 software.

Marcon F., Zuo J., Pearce H., Nicol S., Margielewska-Davies S., Farhat M., Mahon B., Middleton G., Brown R., Roberts K.J, & Moss P. (2020). NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype. Oncoimmunology, 9(1), 1845424.

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Phenotyping NK Cell Receptors

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Analysis of activating receptors on NK cells were performed on PB and BM samples using a gating strategy on CD138^-CD45⁺CD56⁺CD3^-CD16^+/− cell after CD138⁺ cell (corresponding to PCs) gate exclusion (Figure 1A). The samples were stained with anti-CD138/FITC, anti-CD3/APC-H7, anti-CD56/PE, anti-CD45/PE-Cy7, anti-CD16/PerCP, anti-NKG2D/APC, anti-DNAM-1/FITC (BD Biosciences, San Jose, CA, USA) and anti-NKp30/APC (BioLegend, San Diego, CA, USA) for 25 min at 4 °C [50 (link)]. Expression levels of NK cell receptors on NK cell subsets derived from healthy PB donors are shown in Figure S4.
All the samples were acquired using a FACSCanto II (BD Biosciences, San Jose, CA) and data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).

Vulpis E., Stabile H., Soriani A., Fionda C., Petrucci M.T., Mariggio’ E., Ricciardi M.R., Cippitelli M., Gismondi A., Santoni A, & Zingoni A. (2018). Key Role of the CD56lowCD16low Natural Killer Cell Subset in the Recognition and Killing of Multiple Myeloma Cells. Cancers, 10(12), 473.

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