HSPC-NK cell phenotype was determined by staining for CD56-PE-Cy7 (Biolegend, #318318), NKG2A-APC (Beckman Coulter, #A60797), CD16-FITC (Biolegend, #302006), pan-KIRs-PE (Biolegend, #339506, 312606 and 312708), DNAM-1-FITC (BD, #559788), NKp46-PE (Biolegend, #331908) and NKG2D-APC (Biolegend, #320808) (CellGro vs NK MACS) or CD56-BV510 (Biolegend, #318340), CD45-BV421 (Biolegend, #368522), NKG2A-PE-Cy7 (Beckman Coulter, #B10246), DNAM-1-FITC (Biolegend, #337104), NKp46-PE (Biolegend, #311908), NKp44-PE (Biolegend, #325108), NKp30-APC (Biolegend, #325210) and NKG2D-APC (Biolegend, #320808) (GMP validations runs). Briefly, 200.000 cells were washed using PBS/0.5% BSA and incubated with antibodies in PBS/0.5% BSA at 4 °C for 30 min. Cells were then washed twice with PBS/0.5% BSA and resuspended in PBS/0.5% BSA containing Sytox Blue (1:5000 diluted, invitrogen, #S34857) for CellGro vs NK MACS experiments or 7-AAD (1:1000 diluted, Sigma, #A9400) for GMP validation runs. Cells were acquired on the Gallios (CellGro vs NK MACS) or Navios (GMP validation runs) flowcytometers and analyzed using Kaluza V2.1.3.
Nkg2d apc
Manufactured by BioLegend
Sourced in United States
NKG2D-APC is a monoclonal antibody conjugated with the fluorescent dye APC (Allophycocyanin). It is designed to detect the expression of the NKG2D receptor on the surface of natural killer (NK) cells and certain T cell subsets.
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Lab products found in correlation
Cd56 pe, by BioLegend (3 mentions)
Nkp30 apc, by BioLegend (2 mentions)
Nkg2a apc, by Beckman Coulter (2 mentions)
Nkp30 pe, by BioLegend (2 mentions)
Nkp46 pe, by BioLegend (2 mentions)
Dnam 1 fitc, by BioLegend (2 mentions)
Fitc nkp46, by BioLegend (2 mentions)
Cd16 pe, by BD (2 mentions)
Dnam 1 fitc, by BD (2 mentions)
Sytox blue, by Thermo Fisher Scientific (2 mentions)
Cd3 fitc, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (2 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Cd8 fitc, by BioLegend (1 mentions)
Nkg2a pe, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Kaluza 1, by Beckman Coulter (1 mentions)
Cd56 pe cy5, by BioLegend (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Nkg2a pe cy7, by Beckman Coulter (1 mentions)
10 protocols using nkg2d apc
1
Phenotyping of HSPC-derived NK Cells
2
NK Cell Phenotyping in Mouse and Human
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After cocultivating NK cells and PC cells with different treatments for three days, NK cells were collected and washed twice with PBS, and then, anti-human CD3-PerCP/Cyanine5.5, CD16-PE, CD56-PE, NKG2D-APC, NKp46-PE/Cyanine7, and DNAM-1-FITC antibodies were added (BioLegend, San Diego, CA, USA) according to the instructions. The cells were incubated for 20 min in the dark and washed twice with PBS. For the detection of NK cells in mouse peripheral blood, 100 μl mouse peripheral blood was collected, and anti-mouse CD3-PerCP/Cyanine5.5, NK1.1-PE-Cyanine7, NKG2D-APC, NKp46-FITC, and DNAM-1-PE antibodies were added (BioLegend, San Diego, CA, USA) and incubated at room temperature for 20 minutes in the dark. Then, 2 ml RBC lysis solution was added to each tube, mixed well, and incubated for 15 min. Then, the samples were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. All samples were then analyzed by multicolor flow cytometry (BD, USA).
3
Immunophenotyping of NK Cell Receptors
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Peripheral blood monouclear cells (PBMCs) were isolated from patients and healthy individuals, suspended in RPMI 1640 medium containing 2 mM/L L-glutamine and 10% fetal calf serum (Hyclone). Isolated PBMCs were stained with fluorochrome-conjugated antibodies to CD3-PerCP/cy5.5, CD56-FITC, CD8-FITC and CD4-FITC (Biolegend, San Diego, CA). NK cells surface receptors were stained with fluorochrome-conjugated antibodies to NKp46-PE, NKG2A-PE, NKp30-APC, NKG2C-APC, NKG2D-APC and isotype matched controls (Biolegend, San Diego, CA). Stained PBMC were isolated using a FACS Calibur flow cytometer (Becton Dickinson, USA) and analyzed by FlowJo analysis software (Treestar, Ashland, OR).
4
Multiparametric Flow Cytometry Analysis of Natural Killer Cells
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MNCs were stained with labeled antibodies, CD3 ECD (Biolegend), CD45 Krome Orange (R&D systems), CD56 PE-Cy5 (Biolegend), CD16 APC-Cy7 (Biologend), CD326 PerCPCy5.5 (Biolegend). Phenotypic analysis was performed using DNAM-1 FITC (Becton Dickinson), 2B4 FITC (Biolegend), NKG2A APC (Beckman Coulter), NKG2D APC (Biolegend), NKp30 PE (Biolegend) and NKp46 PE(Biolegend), isotype controls for IgG1 and IgG2a, (all Biolegend). Dead cells were stained with 1:1000 diluted sytox blue (Life Technologies; Invitrogen). Flow cytometry analysis was performed on a Gallios flow cytometer from Beckman Coulter. Analysis was done in Kaluza 1.5 (Beckman Coulter). Gating strategy is shown in Supplementary Figure 1 .
5
Characterization of Primary Human NK Cells
6
Phenotyping of Natural Killer Cells
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Antibodies and other reagents used were: Intracellular Fixation and Permeabilization Buffer kit, fixable viability dye e-Fluor 780, CD56 PerCP-eFluor 710, MICA/B Alexa Fluor 488, CD69 PE-Cy7 (eBioscience, Grand Island, NY, USA); CD16 PE, CD56 PE (BD Biosciences, San Jose, CA, USA); CD3 FITC, IFN-γ PE-Vio770, TNF-α PE-Vio770, CD107a FITC (Miltenyi Biotec, Auburn, CA, USA); CD3 PE-Cy7, CD3 FITC, CD56 APC (MY31) (Tonbo Bioscience, San Diego, CA, USA); perforin Pacific Blue, granzyme B Pacific Blue, NKG2D APC (BioLegend, San Diego, CA, USA); and survivin Alexa Fluor 647 (Cell Signaling Technology, Danvers, MA, USA). Data were collected on a Miltenyi MACSQuant Analyzer flow cytometer (Miltenyi Biotec, Auburn, CA, USA) and analyzed using FlowJo v10.0.8p software (Tree Star, Ashland, OR, USA).
7
Analyzing NK Cell NKG2D Downregulation by SEVs
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To analyze the effect of SEVs on the activity of NK cells, the downregulation of the NKG2D receptor on NK cells was analyzed. Freshly isolated PBMCs were seeded in AIM-V medium (500,000 cells/well) in 96-well plates and treated with 50 µL PBS as vehicle control, 50 µL of SEC-isolated SEVs, 50 µL of UCopt-isolated SEVs, and 50 µL of supernatant from UC (SUP). After 24 h, PBMCs were incubated with FcR-blocking reagent (1:100) for 10 min at 4 °C and stained using CD3-FITC (1:50), CD56-PE (1:50) and NKG2D-APC (1:50) antibodies from BioLegend to measure the expression of the receptor on NK cells (CD3-CD56+).
8
Cytokine Profiling of PBMCs Treated with CM-Exs
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PBMCs were stimulated with CM-Exs (10, 50, or 100 mg/mL) with or without IL-2 (10 4 U/mL) (BD Biosciences, San Jose, CA, USA) and IL-12 (10 4 U/ mL) (BD Biosciences) for 5 h. For intracellular cytokine staining, we added Brefeldin A to the culture medium for the final four hours. Following in vitro stimulation for 5 h, PBMCs were incubated with ViViD dye (LIVE/DEAD Fixable Dead Cell Stains, ThermoFisher Scientific, Waltham, MA, USA) to identify dead cells, followed by surface staining with the following antibodies: CD56-AlexaFluor 488 (BD Biosciences), T cell receptor (TCR) Va24-Ja18-FITC (BioLegend, San Diego, CA, USA), CD16-PE (BD Biosciences), CD1d-tetramer-PE (MBL, Nagoya, Japan), CD8-PerCP-Cy5.5 (BioLegend), CD69-PerCP-Cy5.5 (BD Biosciences), CD4-PE-Cy7 (BD Biosciences), CD16-APC (BD Biosciences), NKG2D-APC (BioLegend), TCR cd-APC (BioLegend), CD3-APC-Cy7 (BioLegend), CD8-V500 (BD Biosciences), and CD3-V500 (BD Biosciences). Cells were then fixed and permeabilised using BD Cytofix/Cytoperm (BD Biosciences) and stained for IFN-c-PE (BioLegend), IL-17-PE (BioLegend), IL-10-Bv421 (BioLegend), and IFN-c-Bv421 (BD Biosciences). FACS analysis was performed using a FACSVerse (BD Biosciences) with FlowJo software (BD Biosciences).
9
Phenotypic and Functional Analysis of Immune Cells
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The antibodies (FITC-CD3, PE-CD56, APC-NKG2D, BV421-CD8, and APC-MICA/B) and their respective isotype antibodies were purchased from BioLegend (San Diego, CA, USA). The Annexin V-FITC -7AAD (7-Amino-Actinomycin D) kit was also purchased from BioLegend (San Diego, CA, USA). The HSP90 inhibitors 17-DMAG and ganetespib (STA-9090) were purchased from Selleckchem (Boston, MA, USA). These HSP90 inhibitors were dissolved in DMSO and stored at −80 °C at a concentration of 50 mM (please note that the control DMSO concentration of 17-DMAG was 0.1‰, and that the control DMSO concentration of ganetespib was 0.02‰). The FxCycle™ Violet stain and CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) were used to distinguish tumor cells from CIK cells using flow cytometry. Hoechst 34580 (Merck, Sigma, Darmstadt, Germany) was added prior to flow cytometry to stain the dead cells. The CellEvent Tm Caspase-3/7 Green flow cytometry assay kit was purchased from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA).
10
Multiparametric Flow Cytometry Analysis of Murine Splenocytes
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An LSR Fortessa (BD Biosciences, Franklin Lakes, NJ) was used for multiparametric flow cytometry analysis. A Live/Dead Fixable Blue Dead Cell Stain Kit (Molecular Probes; Thermo Fisher Scientific, Grand Island, NY) was used to exclude dead cells. The following murine monoclonal antibodies (mAbs) were used to stain Balb/c splenocytes to characterize immune cell subsets: FITC-CD8 (Clone 53-6.7; BD Biosciences), PE-CD49b (Clone DX5; BioLegend, San Diego, CA), PE-PD-L1 (Clone 10F.9G2; BioLegend), PerCP-Cy5.5-B7-1 (Clone 16-10A1; BioLegend), PerCP-Cy5.5-FOXP3 (Clone FJK-16s; eBioscience, San Diego, CA), PE-Cy7-CD122 (Clone Tm-b1; eBioscience), APC-NKG2D (Clone CX5; BioLegend), APC-Cy7-CD3 (Clone 17A2; BioLegend), Pacific Blue-CD44 (Clone IM7; BioLegend), AF700-CD4 (Clone GK1.5; eBioscience), BV605-CD19 (Clone 6D5; BioLegend), BV605-PD-1 (Clone 29F.1A12; BioLegend), BV510-NKp46 (Clone 29A1.4; BioLegend), Pacific Blue-CD27 (Clone LG.3A10; BioLegend), FITC-CD11b (Clone M1/70; BD Biosciences), AF700-CD11b (Clone M1/70; BioLegend), APC-Cy7-CD11c (Clone N418; BioLegend), BV510-CD3 (Clone 17A2; BioLegend), Pacific Blue-Ly6-G (Clone 1A8; BioLegend), BV605-Ly6-C (Clone HK1.4; BioLegend).
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