Brilliant violet 421 anti mouse f4 80

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Brilliant Violet 421 anti-mouse F4/80 is a fluorochrome-conjugated antibody that binds to the F4/80 antigen expressed on mouse macrophages.

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F4/80 (349 citations) Anti-F4/80 (96 citations) Brilliant Violet 421 (66 citations) Anti-mouse F4/80 (25 citations) F4/80-Brilliant Violet 421™ (5 citations) Anti-F4/80-Brilliant Violet 421 (2 citations) Brilliant Violet 421™ anti-mouse F4/80 antibody (2 citations) Brilliant Violet 421 anti‐mouse F4/80 BM8 (2 citations)

5 protocols using brilliant violet 421 anti mouse f4 80

Comprehensive Immunological Profiling of Tumor Cell Lines

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4T1, H22, HepG2, NIH‐3T3, and Panc02 were purchased from BeNa Culture Collection, China. Anti‐mouse PD‐1 antibody (PD‐1 Ab, BE0146‐100 mg) was purchased from Bio X Cell. Anti‐human PD‐1 Ab was obtained as a gift from Livzon Pharmaceutical Group Inc., China. Flow cytometry antibodies included PE anti‐mouse CD274 (B7‐H1, PD‐L1) (124308), APC/Cyanine7 anti‐mouse CD45 (103132), FITC anti‐mouse CD3 (100204), Brilliant Violet 421 anti‐mouse CD4 (100563), PE/Cy7 anti‐mouse CD8a (100722), PE anti‐mouse IFN‐γ (505808), Alexa Fluor 647 anti‐human/mouse Granzyme B (515406), APC anti‐mouse Ki‐67 (652406), PE anti‐mouse/human CD44 (103008), PE anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) (108407), APC/Cyanine7 anti‐mouse/human CD11b (101226), Brilliant Violet 421 anti‐mouse F4/80 (123137), PE anti‐mouse CD86 (105008), PE/Cy7 anti‐mouse CD206 (MMR) (141720), Alexa Fluor 647 anti‐mouse FOXP3 (126408), APC anti‐mouse CD49b (pan‐NK cells) (108910), PE anti‐mouse CD25 (102008), PE anti‐mouse/human CD44 (103008), APC anti‐mouse CD62L (104412), Zombie Aqua Fixable Viability Kit (423102), Cell Activation Cocktail (with Brefeldin A) (423304), True‐Nuclear Transcription Factor Buffer Set (424401), and Intracellular Staining Permeabilization Wash Buffer (10X) (421002) were purchased from BioLegend.

Liu X., Ye N., Liu S., Guan J., Deng Q., Zhang Z., Xiao C., Ding Z., Zhang B., Chen X., Li Z, & Yang X. (2021). Hyperbaric Oxygen Boosts PD‐1 Antibody Delivery and T Cell Infiltration for Augmented Immune Responses Against Solid Tumors. Advanced Science, 8(15), 2100233.

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Lung Cell Dissociation and Phenotyping

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Whole lungs were dissociated into single‐cell suspensions using the gentleMACS Dissociator. Red blood cell lysing buffer (Sigma‐Aldrich) was used for red cell lysis.
Lung cells were blocked with anti‐mouse CD16/32 (101319; Biolegend) and then stained with antibodies PerCP anti‐mouse/human CD11b (101229; Biolegend), Brilliant Violet 421 anti‐mouse F4/80 (123137; Biolegend), APC/Cy7 anti‐mouse CD45 (103116; Biolegend), PerCP/Cy5.5 anti‐mouse CD11c (117328; Biolegend), PE Siglec‐F (552126; BD Biosciences), PerCP/Cy5.5 anti‐mouse CD4 (100540; Biolegend), PE/Cy7 anti‐mouse CD3ε (100320; Biolegend), Brilliant Violet 421 anti‐mouse CD335 (NKp46) (137612; Biolegend), APC anti‐mouse CD8a (100712; Biolegend), Brilliant Violet 421 anti‐mouse Ly‐6G/Ly‐6C (Gr1) (108433; Biolegend) and Zombie Aqua Fixable Viability Kit (423102; Biolegend). Flow cytometric data acquisition was performed on BD FACS Canto II machine and data analysis was performed using FlowJo software.
Gating for CD45+AquaZombie−Siglec‐F+CD11c+Gr1− cells was used for AMs; CD45+AquaZombie−F4/80+CD11b+Gr1− for IMs CD45+AquaZombie−NKp46+ for natural killer (NK) cells, CD45+AquaZombie−CD3+CD8+ for CD8 T cells.

Alikhanyan K., Chen Y., Kraut S, & Sotillo R. (2020). Targeting alveolar macrophages shows better treatment response than deletion of interstitial macrophages in EGFR mutant lung adenocarcinoma. Immunity, Inflammation and Disease, 8(2), 181-187.

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Macrophage Isolation and Characterization

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Naive macrophages were detached from non-tissue culture-treated plates after 7 days differentiation in M-CSF-containing media by scraping in versene. Cells were pelleted (1000 g, room temperature, 5 min), washed in FACS buffer [1% (w/v) bovine serum albumin in phosphate buffered saline (PBS)] and then kept at 4°C for subsequent steps. Cells were resuspended and incubated with 10 µg/ml anti-CD16/32 Fc block (BD Bioscience) in FACS buffer for 15 min, prior to incubation for 30 min with 1 µg/ml Brilliant Violet 421 anti-mouse F4/80 and 1 µg/ml PerCP/Cy5.5 anti-mouse/human CD11b (Biolegend) in FACS buffer. Cells were washed in FACS buffer and fixed by incubation with 4% formaldehyde in PBS for 20 min. Fixed cells were washed and resuspended in FACS buffer prior to analysis using FACS Canto (Becton Dickinson).

Darling N.J., Toth R., Arthur J.S, & Clark K. (2017). Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages. Biochemical Journal, 474(4), 521-537.

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Cell Purity Assessment via Flow Cytometry

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To assess purity of cells, macrophages were dissociated from tissue culture dishes using a non-enzymatic CellStripper Dissociation Reagent (Corning), blocked with anti-mouse CD16/32 (BioLegend), stained with Brilliant Violet 421 anti-mouse F4/80 (BioLegend) and Alexa Fluor 488 anti-mouse/human CD11b (BioLegend) or Zombie Red Fixable Viability Dye (BioLegend), fixed with 4% paraformaldehyde (BioLegend), and analyzed using a MACSQuant VYB (Miltenyi Biotec). Analysis of data was performed using FlowJo software (Becton, Dickson & Company).

Chen S., Fuller K.K., Dunlap J.C, & Loros J.J. (2020). A Pro- and Anti-inflammatory Axis Modulates the Macrophage Circadian Clock. Frontiers in Immunology, 11, 867.

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Phenotypic Analysis of Immune Cells

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Single-cell suspension was prepared from mouse popliteal lymph nodes and synovial tissues or retrieved from cell cultures. Cell surface antigens were stained for 30 min on ice with an antibody staining mix diluted in PBS containing 1% BSA. Intracellular staining was performed using the Transcription Factor Buffer Set (562574; BD Biosciences, San Diego, CA, USA) according to the manufacturers’ instructions. The following antibodies were used for the studies: PE anti-mouse F4/80 (123110), Brilliant Violet 421 anti-mouse F4/80 (123131), PE/Cy7 anti-mouse CD45 (157205), PE/Cy7 anti-mouse CD206 (141720), PE/Cy7 anti-mouse CD86 (105014), Brilliant Violet 421 anti-mouse CD86 (105031), FITC anti-mouse CX3CR1 (149019), 7-AAD viability staining solution (420403), and PE/Cy7 rat IgG2a, κ isotype ctrl antibody (400521) from Biolegend (San Diego, CA, USA); and PerCP-Cy5.5 rat anti-CD11b (550993) from BD Biosciences (San Diego, CA, USA). Cytometry data were acquired on an Attune NxT (Thermo Fisher Scientific) and analyzed using the FlowJo software version 10. Gating strategies are provided in

Supplementary Figure 1

Chen L., Zhou Q., Fang X., Xu Q., Zou Y, & Zhang J. (2024). Administration of Liposomal-Based Pde3b Gene Therapy Protects Mice Against Collagen-Induced Rheumatoid Arthritis via Modulating Macrophage Polarization. International Journal of Nanomedicine, 19, 4411-4427.

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