All participants donated 10 ml of peripheral blood anticoagulated with EDTA, and samples were processed within three hours after extraction. Briefly, 150 μl of peripheral blood was incubated with CD56-Pacific blue (362520), CD16-BrilliantViolet510 (302048), CD19-PercPCy5.5 (302230), NKG2A-APC fyre (375116), NKG2C-PE (375004), NKG2D-FITC (320820) and NKp44-APC (325110) from Biolegend (San Diego, CA, USA) and CD3-ECD (A07748) from Beckman Coulter (Brea, CA, USA), according to manufacturer’s protocols. After monoclonal incubation, red blood cells were lysed with BD FACS lysing solution (349202, Becton Dickinson, Franklin Lakes, NJ, USA). Samples were washed and suspended in 300 μl phosphate saline buffer, and acquired in a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA). For every variable, all files were collected and data was analyzed at the same time with Kaluza v2.1 (Beckman Coulter, Brea, CA) by a blinded operator. NKbright, NKdim and NKT populations were gated by expression of CD56, CD16 and CD3. Subpopulations expressing NKG2A, NKG2C and NKG2D were selected, and NKp44 expression was analyzed in every subpopulation with two different gating strategies to confirm the differences. Gating strategies are detailed in Supplementary File
Cd16 brilliantviolet510
Manufactured by BioLegend
Sourced in United States
CD16-BrilliantViolet510 is a fluorophore-conjugated antibody that binds to the CD16 receptor on the surface of cells. This product can be used for flow cytometry applications to identify and quantify CD16-expressing cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Facs lysing solution, by BD (1 mentions)
Cd3 ecd, by Beckman Coulter (1 mentions)
Cd19 percp cy5, by BioLegend (1 mentions)
Nkg2c pe, by BioLegend (1 mentions)
Nkg2d fitc, by BioLegend (1 mentions)
Nkp44 apc, by BioLegend (1 mentions)
Kaluza v2, by Beckman Coulter (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Granzyme b af647, by BD (1 mentions)
Cd45 apc cyanine7, by BioLegend (1 mentions)
Cd56 brilliant violet 605, by BioLegend (1 mentions)
Cd3 brilliant violet 785, by BioLegend (1 mentions)
Ifn γ fitc, by BioLegend (1 mentions)
Cd33 pe, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Cd19 pe dazzle 594, by BioLegend (1 mentions)
Attune nxt acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Kaluza version 2, by Beckman Coulter (1 mentions)
Cd26 pe, by BioLegend (1 mentions)
Cd4 brilliant violet 605, by BioLegend (1 mentions)
3 protocols using cd16 brilliantviolet510
1
NK Cell Immunophenotyping in Peripheral Blood
2
Phenotypic analysis of immune cells
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Cells were phenotypically analysed using the Beckman Flow Cytometer. To exclude dead cells from the analysis, 7-amino-actinomycin-D was added to the cells prior to acquisition. For 9-color analyses on the BACKMAN, the following conjugated mAbs were combined: CD56-Brilliant Violet 605 (catalog number: 362538, BioLegend, China), CD16-Brilliant Violet 510 (catalog number: 302048, BioLegend, China), CD45-APC-Cyanine 7 (catalog number: 368516, BioLegend, China), CD3-Brilliant Violet 785 (catalog number: 344842, BioLegend, China), NKG2A-FITC (catalog number: 130-113-565, BioLegend, Miltenyi, Germany), CD8-PB450 (catalog number: 562428, BD, USA), CD4-APC (catalog number: 5553492, BD, USA), CD33-PE (catalog number: 555450, BD, USA), IFN-γ-FITC (catalog number: 506504, BioLegend, China), CD107a-PE-Cyanine 7 (catalog number: 561348, BD, USA), and Granzyme B-AF647 (catalog number: 560212, BD, USA). Before 9-color analyses were performed, all conjugates were titrated and individually tested for sensitivity, resolution, and compensation of spectral overlap. Isotype controls were used to define marker settings. The combinations were balanced in fluorochrome combinations to avoid antibody interactions and steric hindrance and to detect dimly expressing populations. Then, the cells were analysed using FlowJo software 10.
3
Flow Cytometric Leukocyte Profiling
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Flow cytometric characterization of leukocytes was performed as previously established (25 (link)). Within 1 h of collection, 100 µl of EDTA-anticoagulated whole blood was added to a master mix consisting of 100 µl fluorescence-activated cell scanning (FACS) staining buffer (BioLegend, San Diego, CA, USA) and 2 µl of each of the following antibodies: anti-human CD14-Pacific Blue™, CD16-Brilliant Violet 510™, CD4-Brilliant Violet 605™, CD45-Brilliant Violet 711™, CD3-Alexa Fluor 488™, CD26-PE, CD19-PE/Dazzle 594™, CD8-PE/Cyanine7, and CD41-Alexa Fluor 647™ (all BioLegend, San Diego, CA, USA). The mixture was incubated in the dark at room temperature for 30 min. Stained samples were fixed by adding 800 µl of 0.5% paraformaldehyde in phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) and kept at +4°C in the dark. Samples were acquired on an Attune NxT Acoustic Focusing Cytometer (ThermoFisherScientific, Waltham, MA, USA) within 3 days. Stability of signal detection was verified and documented by daily measurements of Attune Performance tracking beads. Kaluza version 2.1 software (Beckman Coulter, Brea, CA, USA) was used for gating. Scatter parameters were interpreted as measures of morphological features, i.e., side scatter as a measure of granularity and forward scatter as a measure of cell size.
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