The anti-human mAbs used were as follows: PE-CD274, PE-EGFR, PE-CD3, PE-CD226 (DNAM-1), PerCP-Cy5.5-NKG2D, BV421-NKp30, BV510-granzyme B, PE-Cy5-CD107a, and PE-Smad2 (pS465/pS467)/Smad3 (pS423/pS425) (BD Biosciences, San Jose, CA); APC-CD56 (BioLegend, San Diego, CA); PE-Cy7-perforin (eBioscience, San Diego, CA), and PE-TGFβ receptor II (R&D Systems). Samples were acquired on a FACSCalibur flow cytometer or FACSVerse (Becton Dickinson, Franklin Lakes, NJ), and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR). Isotype control staining was < 5% for all samples analyzed. For human PBMC subset analysis, PBMCs from three healthy donors were obtained from the NIH Clinical Center Blood Bank (NCT00001846), as previously described (26 (link)). Frozen PBMCs were thawed and seeded at 5×106 per well, and treated with IL-15SA/IL-15RA at 0, 1, 5, 25, and 50 ng/ml for 72h. Cells were harvested and assessed for the frequency of immune cell subsets by multi-parametric flow cytometry as previously described (27 (link)).
Percp cy5.5 nkg2d
Manufactured by BD
PerCP-Cy5.5-NKG2D is a fluorophore-conjugated antibody that binds to the NKG2D protein, which is expressed on the surface of natural killer (NK) cells and certain T cells. This product can be used in flow cytometry applications to detect and quantify NKG2D-expressing cells.
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Lab products found in correlation
Pe cy7 perforin, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Cd56 apc, by BioLegend (2 mentions)
Egfr pe, by BD (2 mentions)
Facsverse, by BD (2 mentions)
Cd3 pe, by BD (1 mentions)
Cd107a pe cy5, by BD (1 mentions)
Pe cy7 nkp46, by Thermo Fisher Scientific (1 mentions)
Ifn γ pe cy7, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
Cd56 apc, by Thermo Fisher Scientific (1 mentions)
Cd3 percp, by Thermo Fisher Scientific (1 mentions)
Cd107a fitc, by BD (1 mentions)
3 protocols using percp cy5.5 nkg2d
1
Phenotypic Analysis of PBMC Subsets
2
Multiparameter Flow Cytometry Assay
3
Multiparameter Flow Cytometry of NK Cells
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Aliquots of 1 × 106 NK cells were analyzed for surface markers using a panel of prediluted fluorochrome-conjugated anti-human monoclonal antibodies. FITC-CD3, PE-Cy™7-IFNγ, FITC-CD107a, PE-NKp44, and PerCP-Cy™5.5-NKG2D were purchased from BD Biosciences. PerCP-CD3, PE-CD253/TRAIL, Alexa Fluor-700-TNFα, APC-CD56, APC-NKp30, PE-Cy7-NKp46, and Alexa Fluor-700-TIM-3 were purchased from eBioscience (San Diego, CA, USA). PE-Galectin-9 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Samples were run on a FACSAria II dual-laser 8-color cytometer (BD Biosciences, Europe), and data were analyzed with FACSDiva software 6.1.3.
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