Tumors were disaggregated and digested in collagenase D and DNase for 30 min at 37°C to obtain a single-cell suspension. After neutralizing unspecific binding with a CD16/CD32 antibody (clone 93), single-cell suspensions were stained with fixable Live/Dead staining (Life Technologies), followed by incubation with specific monoclonal antibodies diluted 1:100 (primary antibodies directly conjugated) to assess the phenotype. The antibodies used were: CD45 (REA737, Miltenyi Biotec); Ly-6G (1A8, Biolegend); Ly6C (REA796, Miltenyi Biotec), CD11b (REA592, Miltenyi Biotec); F4/80 (REA126, Miltenyi Biotec), CD19 (REA749, Miltenyi Biotec), CD206 (C068C2, Biolegend), CD11c (REA754, Miltenyi Biotec), B220 (RA3-6B2, Biolegend), CD3 (REA641, Miltenyi Biotec), CD8 (REA601, Miltenyi Biotec), CD4 (REA604, Miltenyi Biotec), NK1.1 (REA1162, Miltenyi Biotec), NKp44 (P44-8 BioLegend), NKp46 (29A1.4, BD). For flow gating, we used isotype controls of fluorescence minus one control. All the antibodies were purchased from Miltenyi Biotec or Biolegend. Samples were acquired on a BD Canto-II flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR). The respective RRID of each reagent is listed in the key resources table .
Nkp44
Manufactured by BioLegend
Sourced in United States
NKp44 is a cell surface receptor expressed on natural killer (NK) cells. It is a member of the natural cytotoxicity receptor (NCR) family and plays a role in the activation and function of NK cells.
Automatically generated - may contain errors
Lab products found in correlation
Nkp30, by BioLegend (2 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Cd11b, by Miltenyi Biotec (1 mentions)
F4 80, by Miltenyi Biotec (1 mentions)
Live dead staining, by Thermo Fisher Scientific (1 mentions)
Ly6g 1a8, by BioLegend (1 mentions)
Cd206, by BioLegend (1 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)
Pd l1, by BioLegend (1 mentions)
Cd112, by BioLegend (1 mentions)
Tocilizumab actemra, by Roche (1 mentions)
Ulbp1, by R&D Systems (1 mentions)
Cd226, by BioLegend (1 mentions)
6 protocols using nkp44
1
Tumor Immune Cell Phenotyping
2
Isolating Oral Cancer Cells and Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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)).
3
Peripheral Blood Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Clinical Chemistry laboratory of Karolinska University Hospital measured the absolute numbers of leukocytes, neutrophils, eosinophils, basophils, and monocytes per liter of peripheral blood using XE Sysmex flow cytometry-based analysis. Peripheral blood mononuclear cells (PBMC) were stained freshly using the following antibodies (clones): CD45RA (B56), TcRgd (B1), HLA-DR (L43), CD4 (OKT4), CD138 (ID4 or DL-101), CD19 (HIB19), NKp44 (P44-8), CD16 (3G8), CD69 (FN50), CD28 (CD28.2), CD45 (HI30), IL21R (2G1-K12), TREM-1 (TREM-26) all from Biolegend, CD3 (UCHT1) and NKG2A (Z199.1) from Beckman Coulter, IgD (IA6-2), CD14 (Mphi 9), CD27 (M-T271), CD56 (BI59) from Beckton Dickinson, NKG2D (1D11) from eBioscience. The definition of major cell populations was as described in35 (link), and performed at the Division of Rheumatology in the Center for Molecular Medicine, Karolinska Institutet.
4
Multiparameter Analysis of Immune Checkpoint Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
5
Phenotyping CXCL10-Expressing Cells in Liver Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To phenotype the cells expressing CXCL10, fluorescence microscopy was performed using an RNAscope approach with a probe targeting CXCL10 (#311851, ACD, Newark, CA), combined with immunofluorescence using nuclear stain (DAPI) with antibodies to either CD4 (Rabbit; 1:100, Abcam), CD8 (Rabbit; 1:100, Thermo Fisher), NKp44 (Mouse; 1:500, Biolegend, San Diego, CA) with NKp46 (Mouse; 1:500, R&D Systems, Minneapolis, MN) and CD57 (Mouse; 1:100 Invitrogen (Carlsbad, CA) for natural killer cells, CD68 (Mouse; 1:500, Biocare, Pacheco, CA) with CD163 (Mouse; 1:500, Novo Castra, Newcastle Upon Tyne, UK) for myeloid cells or Hepatocyte monoclonal antibody (MA5-12417-OCH1E5, Thermo Fisher) for hepatocytes. Up to four sections of 5μm each were screened and using a confocal microscope (Olympus, Fluoview FV10i), representative pictures of CXCL10+ cells were taken to evaluate the type of cells expressing CXCL10 in liver biopsy specimens.
6
Flow Cytometry Analysis of NK Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK cells were defined by being positive for anti-CD56-PE (BD Biosciences) and negative for CD3 staining. FACS staining was performed using conjugated antibodies against PILRα (R&D systems), NKp44 (BioLegend) and NKp46 (R&D systems). Binding of antibodies against PANP (Abgent), Collectin12 (R&D systems) and NTB-A (BioLegend) were detected using the compatible secondary antibodies; Alexa Fluor 647-conjugated AffiniPure donkey anti-rabbit IgG, Alexa Fluor 647-conjugated AffiniPure donkey anti-goat IgG and Alexa Fluor 647-conjugated AffiniPure goat anti-mouse IgG (all purchased from Jackson ImmunoResearch). The anti-HA1 mAb (H17-L2) was a kind gifts from Jonathan Yewdell, National Institutes of Health. Biotinylated-MALII (vector laboratories) was detected using streptavidin APC antibody (BioLegend). For the fusion proteins detection, a secondary antibody staining of anti-human APC (Jackson ImmunoResearch) was used. All staining were analyzed by flow cytometry using the CellQuest software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!