For cellular surface staining, cells were labelled with antiCD4-FITC, antiCD8-PE, antiNK1.1-PE, antiLy6G-Pacific Blue, antiCD11b-PE (BD Biosciences) or antiCD11c-FITC (Miltenyi Biotec) diluted in RPMI 1640 culture medium with 10% foetal calf serum (FCS). To determine GZMA-positive populations, cells were fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) following manufacturer instructions. Cells were stained with antiGZMA antibody diluted 1:100 (provided by Markus Simon [15 (link)]), followed by incubation with a secondary antibody APC-conjugated anti-rabbit IgG (eBiosciences). To analyze IFNγ-producing cells, these were incubated overnight with Purified-Protein Derivative (PPD) 10μg/ml (Statens Serum Institute, SSI). Golgi inhibitor GolgiPlug (BD Biosciences) was added to cells during the last six hours of incubation. Cells were then fixed and permeabilized with the Cytofix/Cytoperm Fixation/Permeabilization Kit and stained with anti-IFNγ-APC (BD Biosciences).
Anti nk1.1 pe
Manufactured by BD
Sourced in United States
Anti-NK1.1-PE is a fluorescently labeled antibody that binds to the NK1.1 antigen, which is expressed on natural killer cells and a subset of T cells in mice. This product can be used for the identification and enumeration of these cell populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b pe, by BD (2 mentions)
Anti cd11b apc cy7, by BD (2 mentions)
Anti cd11c pe, by BD (2 mentions)
Anti ifn γ apc, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Purified protein derivative, by Statens Serum Institut (1 mentions)
Anti cd8 pe, by BD (1 mentions)
Anti cd4 fitc, by BD (1 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd206 pe, by BioLegend (1 mentions)
Anti nk1 1 percp cy5, by BD (1 mentions)
Anti cd11b percp cy5, by BD (1 mentions)
Anti cd8a fitc, by BD (1 mentions)
Anti cd11c apc, by Thermo Fisher Scientific (1 mentions)
Anti cd4 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd16 cd32 antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd45 apc cy7, by BD (1 mentions)
Anti cd4 pe cy7, by BioLegend (1 mentions)
Anti cd3e percp cy5, by Thermo Fisher Scientific (1 mentions)
Anti cd68 pe, by BioLegend (1 mentions)
5 protocols using anti nk1.1 pe
1
Multiparametric Flow Cytometry for Immune Profiling
2
Isolation and Characterization of Immune Cells from Mouse Spinal Cord
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spinal cord, dissected from mice transcardially perfused with PBS, was minced into 1 mm3 pieces in solution containing 1 mg/mL or 0.1 mg/mL collagenase IV (Worthington Biochemical Corporation, USA) plus 0.4 mg/mL DNase I (Roche, Switzerland), and incubated at 37 °C for 15 min. For isolation of immune cells and microglia, cells were resuspended in 37% Percoll (GE Healthcare, USA) and centrifuged at 780 × g for 20 min. After centrifugation, myelin debris was removed and the cell pellet was collected. Cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then stained with combinations of the following antibodies: anti-CD45-PE-Cy7, anti-CD45-APC-Cy7, anti-CD4-PE, anti-CD8a-FITC, anti-CD8a-APC-Cy7, anti-NK1.1-PErCP-Cy5.5, anti-NK1.1-PE, anti-CD11c-PE, anti-CD11b-APC-Cy7, anti-CD11b-PerCP-Cy5.5 (BD Biosciences, USA), anti-CD4-APC, anti-CD3e-PerCP-Cy5.5, anti-CD86-APC, anti-CD25-PE (eBioscience, USA), anti-CD11c-APC, anti-I-A/I-E (MHC class II)-FITC, anti-CD4-PE-Cy7, anti-CD4-APC, anti-CD68-PE, anti-H-2Kb/H-2Db (MHC class I)-PE, anti-CD206-PE, anti-CD178 (FasL)-PE, and Armenian Hamster IgG Isotype control-PE (BioLegend, USA). Flow cytometry was performed by using FACS Aria and FACS Verse flow cytometers (BD Biosciences, USA) and the data was further analyzed using FlowJo Software (FlowJo, LLC, USA).
3
Multiparametric Flow Cytometry of Mouse MNCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse MNCs were stained with the following fluorescence-conjugated surface mAbs: anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE-cy7, anti-NK1.1-PE, anti-CD11b-APC-cy7, anti-CD103-BV421, anti-CD11c-BV510, and anti-CD25-APC antibodies (BD PharMingen, USA). The cells were stained according to the standard procedure of BD PharMingen. Fixation and permeabilization were performed using the Transcription Factor Buffer Set (BD PharMingen, USA), and the cells were then stained with the anti-FoxP3-BV421 intracellular antibodies for 40 min at 4°C. To detect Th1 and Th17 cytokines, MNCs suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum were stimulated with a cell stimulation cocktail (eBioscience, USA) containing phorbol-12-myristate-13-acetate (PMA, 50 ng/mL), ionomycin (1 μg/mL), and monensin (2 μg/mL) at 37°C with 5% CO2 for 6 h, and followed by intracellular staining with fluorescently labeled anti-IFN-γ and anti-IL-17 antibodies (BioLegend, USA) after washing, fixing, and permeabilizing according to the manufacturer's instructions. Isotype IgGs were used as a control. All samples were detected using a BD LSR Fortessa X-20 Flow Cytometry System (BD, USA) and analyzed using the FlowJo software.
4
Splenic Immune Cell Profiling by FACS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were prepared from the spleen samples and stained for FACS (fluorescence activated cell sorting) analysis as previously described (Shinno-Hashimoto et al., 2022 (link); Zhang et al., 2021b (link)). The following antibodies were used for immunofluorescence staining: anti-CD3-FITC (x40, cat# 100305: BioLegnd, San Diego, CA), anti-CD4-allophycocyanin (x100, cat# 17-0042-82: eBioscience, San Diego, CA), anti-CD8a-allophycocyanin (x100, cat# 553035: BD Bioscience), anti-NK1.1-PE (x100, cat# 553165: BD Bioscience), anti-Ly6c-FITC (x100, cat# 553104: BD Bioscience), anti-CD11b-PE (x400 diluted using FACS buffer, cat# 553312: BD Bioscience, Franklin Lakes, NJ), anti-CD11c-PE (x100, cat# 557401: BD Bioscience), anti-Ter119-PE (x40, cat# 12-5921-83: eBioscience, San Diego, CA), anti-F4/80-PE (x40, cat# 12-4801-80: Invitrogen), and anti-B220-PE (x200, cat# 553309: BD Bioscience). The stained cells were analyzed using a FACSCanto II and FlowJo software (BD Bioscience).
5
In Vivo Confocal Imaging of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy imaging was performed as described previously (24 (link)). Mice were anesthetized (s.c.) with ketamine and xylazine (Syntec, 60 mg/kg and 15 mg/kg, respectively). Before surgery mice received a single dose of IgG (12,5 µg/g, Sigma-Aldrich) and after fifteen minutes they received a mixture of the following antibodies or fluorescent probes: anti-aCD31 BV421 (0,15 µg/g, clone 390, BD), anti-F4/80 BV480 (0,05 µg/g, clone T45-2342, BD), anti-Ly6C BV605 (0,1 µg/g, clone AL-21, BD), anti-aLy6G BV711(0,1 µg/g, clone 1A8, BD), anti-CD19 BB515 (0,1 µg/g, clone 1D3, BD), anti-NK1.1 PE (0,06 µg/g, clone PK136, BD) and anti-CD3e APC (0,06 µg/g, clone 145-2C11, BD) - all from BD Biosciences. In neonatal mice, delivery of drugs (via eye venous plexus) and surgery were made with minor modifications. All images were acquired using an inverted Nikon Eclipse Ti coupled to an A1R confocal microscope loaded with a spectral detector and XYZ motorized stage. Different fields of the organ were pictured. Blood flow analyses was done after FITC-albumin injection (12,5 µg/g, Sigma-Aldrich; i.v.) using resonant scanner (no time lapse). Images and videos rendering were made using NIS-Elements AR Analysis 5.41.01(Nikon) and Volocity(6.3) (Perkin Elmer, Waltham, MA).
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!