Seven and 14 days after implant, epididymal fat pads were harvested following euthanasia and washed in ice cold PBS (Sigma). Fat pads were minced, digested in collagenase (Liberase TL, Roche), and passed through a 100 μm filter. The stromal vascular fraction was harvested by centrifugation, washed in MACS buffer (PBS, 0.5 mM EDTA, 30% BSA), and incubated with anti-CD16/32 prior to adding an antibody cocktail against extracellular antigens. The following antibodies were purchased from Biolegend: anti-CD45 clone 30-F11, anti-Ly6G clone 1A8, anti-F4/80 clone BM8, anti-NK1.1 clone PK136, anti-CD19 clone 6D5, anti-CD11b clone M1/70, anti-CD3 clone 17A2, and Trustain fcX (anti-CD16/32) clone 93. The following isotype controls were also purchased from Biolegend: mouse IgG2a clone MOPC-173; rat IgG2a clone RTK2758; rat IgG2b clone RTK4530. After antibody incubation, cells were washed, fixed, and analyzed using a FACS Aria flow cytometer (BD Biosciences). The number of CD45 cells in each flow cytometry sample was calculated using Bang’s labs Flow Cytometry Absolute Count Standard, which was added prior to data acquisition. FlowJo software (Treestar) was utilized to compensate and analyze data. FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is depicted in Figure S3 .
Anti nk1.1 clone pk136
Manufactured by BioLegend
Sourced in United States
Anti-NK1.1 (clone PK136) is a laboratory reagent used for the detection and analysis of natural killer (NK) cells in mouse samples. The antibody specifically binds to the NK1.1 antigen, which is expressed on the surface of murine NK cells. This product can be used in flow cytometry, immunoprecipitation, and other immunoassays to identify and characterize NK cells in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b clone m1 70, by BioLegend (3 mentions)
Anti cd45 clone 30 f11, by BioLegend (2 mentions)
Anti ly6g clone 1a8, by BioLegend (2 mentions)
Anti cd3 clone 17a2, by BioLegend (2 mentions)
Anti cd49b clone dx5, by BioLegend (2 mentions)
Anti f4 80 clone bm8, by BioLegend (1 mentions)
Anti cd19 clone 6d5, by BioLegend (1 mentions)
Trustain fcx anti cd16 32 clone 93, by BioLegend (1 mentions)
Rat igg2a clone rtk2758, by BioLegend (1 mentions)
Rat igg2b clone rtk4530, by BioLegend (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Liberase tl, by Roche (1 mentions)
Fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Anti cd25 clone pc61, by BioLegend (1 mentions)
Anti cd8α clone 53 6, by Thermo Fisher Scientific (1 mentions)
Anti cd69 clone h1.2f3, by Thermo Fisher Scientific (1 mentions)
Anti cd45 clone 30 f11, by Thermo Fisher Scientific (1 mentions)
Anti cd3ε clone 145 2c11, by BioLegend (1 mentions)
Anti pd 1 clone 29f 1a12, by BioLegend (1 mentions)
9 protocols using anti nk1.1 clone pk136
1
Epididymal Fat Pad Characterization
2
Analysis of Tumor-Draining Lymph Nodes
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One day after NIR-PIT, tumor-draining lymph nodes were removed. Single-cell suspension was prepared from the resected lymph nodes by mechanical crushing and filtration (70 μm cell strainer). The cells were stained with the following monoclonal antibodies: anti-CD45 (clone 30-F11, Thermo Fisher Scientific, Waltham, MA, USA), anti-CD3ε (clone 145-2C11, BioLegend, San Diego, CA, USA), anti-CD8α (clone 53–6.7, Thermo Fisher Scientific), anti-CD25 (clone PC61, BioLegend), anti-CD69 (clone H1.2F3, Thermo Fisher Scientific), and anti-NK1.1 (clone PK136, BioLegend). The cells were also stained with Fixable Viability Dye (Thermo Fisher Scientific) in order to exclude the dead cells from the study. The samples were then analyzed with a cell analyzer (FACS Lyrics and FlowJo software). Each cell population was determined as follows: killer T cell = CD45+/CD3+/CD8+; NK cell = CD45+/CD3−/NK1.1+; NKT cell = CD45+/CD3+/NK1.1+.
3
Detailed Immune Cell Profiling
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Phenotype analysis was performed with staining performed at 4 °C for 20 min with the following antibodies: anti‐mouse CD45 (clone 30‐F11; Biolegend, San Diego, CA, USA), anti‐mouse F4/80 (clone BM8; Biolegend), anti‐mouse CD11c (clone N418; Biolegend), anti‐mouse Ly6C (clone HK1.4; Thermo Fisher Scientific); anti‐CD11b (clone M1/70; Biolegend, San Diego, CA, USA), anti‐CD206 (clone C068C2; Biolegend), anti‐Ly6G (clone 1A8; Biolegend), anti‐NK1.1 (clone PK136; Biolegend), anti‐CD314 (clone C7; Biolegend), anti‐CD4 (clone GK1.5; BD Pharmigen, San Jose, CA, USA), anti CD8 (clone 53‐6.7; BD Pharmigen); antiPD‐1 (clone 29F.1A12; Biolegend), anti‐PD‐L1 (clone 10F.9G2; Biolegend). Cells were detected using the Cyan ADP flow cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed with the Summit 4.3 software (Beckman Coulter). Quadrants were set based on isotype control antibody, and cells were gated among total DAPI− cells.
4
Lipid-based Delivery of mRNA Therapeutics
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Lipids 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(EDOPC), DOPE, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide
(MVL5), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and α-galactosyl
ceramide (α-GalCer or KRN700) were purchased from Avanti Polar
Lipids. Sodium acetate buffer solution (3 M, pH 5.2), nuclease-free
water, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.
Tyrosinase-related protein 2 (TRP-2, 180–188) peptide was purchased
from GenScript. The following antibodies were purchased from BD Biosciences:
anti-TCRβ (clone H57-597), anti-CD8a (clone 53–6.7),
anti-CD11b (clone M1/70), anti-CD45R/B220 (clone RA3-6B2), anti-IFN-γ,
and anti-CD45 (clone 30-F11); BioLegend: anti-F4/80 (clone BM8), anti-CD19
(clone B4), anti-CD11c (clone N418), anti-CD69 (clone H1.2F3), anti-MHC-II
(clone M5/114.15.2), and anti-NK1.1 (clone PK136). Anti-CD8 (clone
KT15) and iTAg Tetramer H-2 Kb TRP2 (SVYDFFVWL) were purchased from
MBL International.
Unmodified TRP2-mRNA, enhanced green fluorescence
protein (eGFP)-mRNA, Cy5-mRNA, ovalbumin (OVA)-mRNA, and luciferase
(LUC)-mRNA were purchased from TriLink Biotechnologies. Six to eight
week-old female C57BL/6 mice were purchased from Charles River (Canada).
(EDOPC), DOPE, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide
(MVL5), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and α-galactosyl
ceramide (α-GalCer or KRN700) were purchased from Avanti Polar
Lipids. Sodium acetate buffer solution (3 M, pH 5.2), nuclease-free
water, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.
Tyrosinase-related protein 2 (TRP-2, 180–188) peptide was purchased
from GenScript. The following antibodies were purchased from BD Biosciences:
anti-TCRβ (clone H57-597), anti-CD8a (clone 53–6.7),
anti-CD11b (clone M1/70), anti-CD45R/B220 (clone RA3-6B2), anti-IFN-γ,
and anti-CD45 (clone 30-F11); BioLegend: anti-F4/80 (clone BM8), anti-CD19
(clone B4), anti-CD11c (clone N418), anti-CD69 (clone H1.2F3), anti-MHC-II
(clone M5/114.15.2), and anti-NK1.1 (clone PK136). Anti-CD8 (clone
KT15) and iTAg Tetramer H-2 Kb TRP2 (SVYDFFVWL) were purchased from
MBL International.
Unmodified TRP2-mRNA, enhanced green fluorescence
protein (eGFP)-mRNA, Cy5-mRNA, ovalbumin (OVA)-mRNA, and luciferase
(LUC)-mRNA were purchased from TriLink Biotechnologies. Six to eight
week-old female C57BL/6 mice were purchased from Charles River (Canada).
5
Comprehensive Immune Cell Profiling by Flow Cytometry
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Flow cytometry was performed as previously described Fang et al. (2010 (link)). The following antibodies were used: anti-CD3e (clone 145-2C11, Biolegend), anti-CD11b (clone M1/70, Biolegend), anti-CD19 (clone 1D3, BD Biosciences), anti-CD27 (clone LG.3A10, Biolegend), anti-CD29 (clone HMβ1-1, Biolegend San Diego, CA), anti-CD44 (clone IM7, Biolegend), anti-CD45.1 (clone A20, Biolegend), anti-CD45.2 (clone 104, Biolegend), anti-CD49a (clone HMα1, Biolegend), anti-CD49b (clone DX5, Biolegend), anti-CD105 (clone MJ7/18, Biolegend), anti-CD106 (clone 429, Biolegend), anti-BrDU (clone PRB-1, eBioscience), anti-CXCR3 (clone CXCR3-173, Biolegend), anti-Eomes (clone Dan11mag, eBioscience), anti-KLRG1 (clone 2F1/KLRG1, Biolegend), anti-Ly-6A/E (Sca-1) (clone E13-161.7, Biolegend), anti-Ly49A (clone A1/Ly49A, Biolegend), anti-Ly49C/I (clone 5E6, Biolegend), anti-Ly49D (clone 4E5, Biolegend), anti-Ly49G2 (clone LGL-1, eBioscience), anti-Ly49H (clone 3D10, Biolegend), anti-NK1.1 (clone PK136, Biolegend), NKG2A/C/E (clone 20d5, Biolegend), anti-Tbet (clone 4B10, Biolegend), and anti-TRAIL (clone N2B2, Biolegend). At least 500 000 cells were analyzed by flow cytometry at the Fox Chase Cell Sorting Facility using an LSR II system (BD Biosciences).
6
Tumor-Infiltrating Immune Cell Profiling
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MC38 or B16-OVA tumor–bearing C57BL/6 mice were euthanized, and tumor tissues were harvested and digested with 10 U/mL collagenase I, 400 U/mL collagenase IV, and 30 U/mL DNase (Yuanye Bio-Technology) to generate single-cell suspensions. The following fluorescently labeled antibodies were then used for staining of different markers: anti-CD45 (clone 30-F11, BioLegend), anti-CD4 (clone RM4-5, eBioscience), anti-CD8 (clone 53-6.7, BioLegend), anti-NK1.1 (clone PK136, BioLegend), anti–granzyme B (clone QA16A02, BioLegend), and anti–IFN-γ (clone XMG1.2, BioLegend). For granzyme B and IFN-γ staining, cells were incubated in culture medium containing a cell activation cocktail (catalog 423303, BioLegend) at 37°C for 5 hours and then washed with permeabilization wash buffer (catalog 421002, BioLegend) before being stained with fluorescently labeled antibodies. Data collection and analysis were performed on a flow cytometer (Becton Dickinson).
7
NK Cell Depletion for Tumor Implantation
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Mice were intraperitoneally treated with 4 mg/kg of anti-NK1.1 (clone PK136; Biolegend) 1 day before the subcutaneous implantation of KPC cells and every three days thereafter. Mouse IgG2a (clone MOPC-173, BioLegend) was used as isotype control. Successful depletion of NK cells was confirmed via flow cytometry.
8
Cell Line Transfection and Flow Cytometry
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NIH3T3, CHO-K1, HEK 293T, and RMA cells were grown in IMDM, Ham’s F12, and RPMI medium, respectively, supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine and transfected using the Lipofectamine 2000 reagent (Life Technologies), following the manufacturer’s instructions. The NIH3T3 and CHO-K1 transfectants were maintained in 0.5 mg/ml geneticin, and SCT Dd or SCT Dd-CD4 cell surface expression was regularly assessed by flow cytometry (clones 5-85 and 34-2-12, Abcam and Biolegend, respectively). The transfectants were FACS-sorted (FACSDiva and FACSARIAIIIU cell sorter, BD) for the cell surface expression of the relevant molecule, and stable transfectants were further selected using 0.8 mg/ml geneticin. Protein cell surface expression was detected by flow cytometry (FACSDiva, BD) using anti-NKG2D (clone C7, Biolegend), anti-Ly49A (clone YE1/48.10.6, Biolegend), anti-CD3 (clone 17A2, Biolegend), anti-NK1.1 (clone PK136, Biolegend), anti-NKp46 (clone 29A1.4, Biolegend), anti-CD49b (clone DX5, Biolegend), and anti-H60a (clone 205326, R\&D systems) antibodies.
9
Synthesis and Characterization of Nanoparticle Immunotherapies
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All nanoparticle syntheses were conducted using ultrapure water with a resistivity of 18.2 MΩ cm (Millipore Corporation, Billerica, MA). Potassium hexacyanoferrate(II) trihydrate (MW 422.39; K4[Fe(CN)6]·3H2O), iron (III) chloride hexahydrate (MW 270.3; Fe(Cl)3·6H2O), and citric acid were purchased from Sigma-Aldrich (St Louis, MO, USA) and used as supplied. Acetone and ethanol solutions were obtained from Sigma-Aldrich. Poly(ethylenimine) (PEI, Mw 2000, Mn 1800, 50% w/v in H2O) was purchased from Sigma-Aldrich, and diluted in sodium acetate buffer solution (pH 5.2, Millipore Sigma, Burlington, MA). Murine CpG oligodeoxynucleotide (CpG) TLR9 ligand (ODN 1585; Class A) was purchased from InvivoGen (San Diego, CA, USA). Fluorescent antibodies against B7H3, CD276 (B7H3), CD80, CD86, CD274 (B7h1, PD-L1), I-A/I-E (MHC-II), CD45, CD8a, CD4, NK1.1 were purchased from Biolegend (San Diego, CA, USA). Anti-CTLA-4 (aCTLA-4) antibody (clone 9D9) was purchased from BioXCell (West Lebanon, NH), anti-CD4 depletion antibody (clone GK 1.5), anti-CD8 depletion antibody (clone 53–6.7), and anti-NK1.1 (clone PK136) were purchased from Biolegend (San Diego, CA, USA).
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