Freshly isolated PBMCs were stained with antibodies specific to the cell surface markers of T cells, B cells, and NK lymphocyte, including anti-CD3, CD19, CD20, CD4, CD8, CD16, CD56, NKp46, NKG2D, NKp30, TLR4, DNAM-1, NKG2A, 4–1BB, OX40, ICOS, PD-1, CTLA4, GITR, LAG3, TIGIT, and TIM3 (BioLegend, San Diego, CA, USA). The stained cells were analyzed using a FACSCant II (BD Biosciences, San Jose, CA, USA), and the data were analyzed using the FlowJo software package (ver.10; Tree Star, Ashland, OR, USA).
Nkp30
Manufactured by BioLegend
Sourced in United States
NKp30 is a cell surface receptor expressed on natural killer (NK) cells. It plays a role in the activation and cytotoxic function of NK cells.
Automatically generated - may contain errors
Lab products found in correlation
Dnam 1, by BioLegend (3 mentions)
Facs cant 2, by BD (2 mentions)
Ctla 4, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Tim 3, by BioLegend (2 mentions)
Nkp44, by BioLegend (2 mentions)
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
Mica b, by BioLegend (1 mentions)
M csf, by BioLegend (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Tgfβrii, by R&D Systems (1 mentions)
Goat anti mouse igg, by BioLegend (1 mentions)
Pd l1, by BioLegend (1 mentions)
Cd112, by BioLegend (1 mentions)
Tocilizumab actemra, by Roche (1 mentions)
6 protocols using nkp30
1
Comprehensive lymphocyte profiling by flow cytometry
2
Immunophenotyping of PBMCs
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Isolated PBMCs were stained with antibodies specific to the cell surface markers of NK lymphocytes, T cells, and B cells, including anti-CD3, CD19, CD20, CD4, CD8, CD16, CD56, NKp46, NKG2D, NKp30, TLR4, DNAM-1, NKG2A, 4-1BB, OX40, ICOS, PD-1, CTLA4, GITR, LAG3, TIGIT, and TIM3 (BioLegend, San Diego, CA, United States). The stained cells were analyzed using FACSCant II (BD Biosciences, San Jose, CA, United States), and the data were analyzed using the FlowJo software package (ver. 10; Tree Star, Ashland, OR, United States). In the PBMC gating, CD3+CD56−cells were defined as T cells, CD3−CD56 + cells were defined as NK cells, CD19 + CD20 + cells were defined as B cells, and T cells were further divided into CD4+ T cells and CD8+ cells.
3
Isolating Oral Cancer Cells and Immune Cells
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RPMI 1640 complete medium with 10% fetal bovine serum (FBS) (Gemini Bio-Product) was used for cell cultures. Oral squamous carcinoma cells and oral squamous carcinoma stem-like cells (OSCSCs) were isolated from cancer patients with tongue tumors at UCLA (2 (link), 33 (link)–35 (link)). Alpha-MEM (Life Technologies, CA, USA) with 10% FBS was used for OCs and DCs cultures. M-CSF was purchased from Biolegend (CA, USA) and RANKL, GM-CSF, and IL-4 were purchased from PeproTech (NJ, USA) and rh-IL-2 was obtained from NIH-BRB. Human CD3/CD28 T cell activator was purchased from Stem Cell Technologies.
Antibodies for MHC-I, KIR2, KIR3, CD44, CD54, B7H1, CD16, NKG2D, MICA/B, KLGR1, CD45, CD3/16/56, CD8, CD3, CD4, GL3, NKp40, NKp30, NKp44, NKp46, and CD94 were purchased from Biolegend (San Diego, CA, USA). ULBP 1–6 antibodies were purchased from R&D Systems. Propidium iodide (PI) was purchased from Sigma (St. Louis, MO, USA). sAJ2 was prepared as described previously (35 (link)).
Antibodies for MHC-I, KIR2, KIR3, CD44, CD54, B7H1, CD16, NKG2D, MICA/B, KLGR1, CD45, CD3/16/56, CD8, CD3, CD4, GL3, NKp40, NKp30, NKp44, NKp46, and CD94 were purchased from Biolegend (San Diego, CA, USA). ULBP 1–6 antibodies were purchased from R&D Systems. Propidium iodide (PI) was purchased from Sigma (St. Louis, MO, USA). sAJ2 was prepared as described previously (35 (link)).
4
Phenotypic Characterization of TGF-β DNR Cells
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Cell phenotype, transduction efficiency, activation, and exhaustion of TGF-β DNR transduced cells and their nontransduced counterparts were determined by flow cytometry, using the following cell surface markers: CD3, CD56 (BioLegend, San Diego, CA), TGF-β RII (“wildtype” R&D, Minneapolis, MN), TGF-β RII (“DNR” Cambridge, UK), goat-anti mouse IgG, CD16, NKG2D, DNAM-1, NKp30, NKp46, CCR2, and CX3CR1 (BioLegend, San Diego, CA and BD Biosciencees, Franklin Lakes, NJ). Where reported, MFI was calculated from the geometric mean.
5
Multiparameter Analysis of Immune Checkpoint Markers
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Alexa Fluor® 647 antihuman TIGIT mAb (clone MBSA43) was obtained from BioLegend® (San Diego, CA, USA). The antibodies to evaluate CD155 (Alexa Fluor® 647, clone SKII.4), CD112 (PE, clone R2.525), PDL-1 (PerCP-Cy5.5, clone 29E.2A3), NKp30 (PE, clone P30-15), NKp44 (PE, clone P44-8), NKp46 (PE, clone 9E2), CD226 (PE, clone DX11), and NKG2D (PE, clone 1D11) were obtained from BioLegend® and MICA (PE, clone 159227), MICB (FITC, clone 236511), ULBP1 (PerCP, clone 170818), ULBP2-5-6 (PE, clone 165903), and B7-H6 (APC, clone 875002) from R&D Systems (Minneapolis, MN, USA). In addition, Ultra-LEAF ™ purified antihuman TIGIT antibody (clone A15153A, mouse IgG2a, BioLegend®) and anti-IL6R antibody (Tocilizumab/Actemra®; Roche, Basil, Switzerland) were used for functional blocking assays at 50 μg/mL and 10 μg/mL, respectively. Stattic (STAT-3 inhibitor; CAS 19983-44-9) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). This reagent was reconstituted in dimethyl sulfoxide (DMSO, Sigma-Merck, Darmstadt, Germany) as 50 mM stock solutions and stored at −20 °C until use.
6
Antibody-mediated NK cell cytotoxicity against infected cells
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Target A549 cells were mock infected or infected at an MOI of 5, and at 16 hpi were incubated for 1 hour with a 1:500 dilution of mouse monoclonal antibodies 1b or 4b against hemagglutinin-neuraminidase (HN) glycoprotein (a kind gift from Dr Randall, University of St Andrews).34 (link) After co-culturing with PM21-NK cells, cytotoxicity was measured by CytoTox-Glo assays as described above.
For experiments blocking NK cell receptors, NK cells were incubated with 10 µg/mL unconjugated antibodies to NK cell receptors NKp30, NKG2D, NKp44 or NKp46 (BioLegend) for 1 hour at 37°C before incubation with NLR-A549 cells that were mock infected or infected at an MOI of 5 with the P/V mutant virus. The cytotoxicity assays were performed on IncuCyte instrument as described above.
For experiments blocking NK cell receptors, NK cells were incubated with 10 µg/mL unconjugated antibodies to NK cell receptors NKp30, NKG2D, NKp44 or NKp46 (BioLegend) for 1 hour at 37°C before incubation with NLR-A549 cells that were mock infected or infected at an MOI of 5 with the P/V mutant virus. The cytotoxicity assays were performed on IncuCyte instrument as described above.
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