After cultivation/polarization, macrophages were detached with flow cytometry buffer (PBS pH 7.4, SERVA; 47302.03), 0.5% (w/v) bovine serum albumin (AppliChem; A1391), 2 mM EDTA (AppliChem; A2937), 0.1% sodium azide (Merck, S2002) with additional 0.4% (w/v) lidocaine (Sigma Aldrich, L7757) and transferred into to a fresh tube. After centrifugation (20°C, 10 min, 400 g), the supernatant was discarded and PM were stained with Zombie Aqua™ (BioLegend; 423101) before blocking non‐specific antibody binding using rabbit serum (Fisher Scientific; 11829230). Subsequently, PM were stained in flow cytometry buffer with the following antibodies: FITC anti‐mouse CD11b (BioLegend; 101206), APC anti‐mouse F4/80 (BioLegend; 123116), PE/Cyanine7 anti‐mouse CD54 (BioLegend; 116122), PerCP anti‐mouse CD86 (BioLegend; 400530), PE anti‐mouse CD200R (BioLegend; 1239089), and Brilliant Violet 421™ anti‐mouse CD206 (BioLegend; 141717). Surface markers were detected using BD LSR Fortessa (BD Biosciences) according to an established protocol. Data were analyzed with FlowJo 10.8 software (BD Biosciences).
Brilliant violet 421 anti mouse cd206
Manufactured by BioLegend
Brilliant Violet 421 anti-mouse CD206 is a fluorochrome-conjugated antibody that binds to the mouse CD206 antigen. CD206, also known as the mannose receptor, is a C-type lectin expressed on the surface of various cell types, including macrophages and dendritic cells. The Brilliant Violet 421 fluorochrome provides a bright signal for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Apc anti mouse f4 80, by BioLegend (2 mentions)
Zombie aqua, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Bovine serum albumin (bsa), by AppliChem (1 mentions)
Lidocaine, by Merck Group (1 mentions)
Fitc anti mouse cd11b, by BioLegend (1 mentions)
Percp anti mouse cd86, by BioLegend (1 mentions)
Ethylene diamine tetraacetic acid (edta), by AppliChem (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Fitc anti human cd206, by BioLegend (1 mentions)
Dil oxldl, by Thermo Fisher Scientific (1 mentions)
Ghost dye violet 510 viability dye, by Cell Signaling Technology (1 mentions)
Pe anti mouse human cd11b m1 70, by BioLegend (1 mentions)
Pe anti human cd36, by BioLegend (1 mentions)
Fitc antimouse cd86 gl1, by BD (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Cd16 32, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
4 protocols using brilliant violet 421 anti mouse cd206
1
Phenotypic Analysis of Polarized Macrophages
2
Characterizing Differentiated Macrophage Phenotypes
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For characterization of differentiated and polarized macrophages, dead cells were first excluded by live/dead staining (Ghost Dye Violet 510 Viability Dye, Cell Signaling Technology) and single cells were gated by plotting the height against the area of FSC. Cells were single stained with the following antibodies: PE anti-mouse/human CD11b (M1/70; BioLegend), APC anti-human CD163 (GHI/61; BioLegend), FITC anti-human CD197 (G043H7; BioLegend), FITC anti-human CD206 (15–2; BioLegend), PE anti-human CD36 (5–271, BioLegend), FITC anti-mouse CD86 (GL1; BD), Brilliant Violet 421 anti-mouse CD206 (C068C2, BioLegend). The percentage of positive cells and the median of fluorescence intensity were quantified by flow cytometry using FACS Canto II (BD). A minimum of 30,000 cells was assessed. Data were acquired and analyzed using FACSDiva software (BD).
To measure the incorporation Dil-labeled oxidized LDL (oxLDL) by M1- and M2a-like macrophages, cells (1–2 × 105) were cultured for 24 h in HBSS supplemented with 0.3% bovine serum albumin in the presence or absence of 1000 ng/ml NETs. Next, cells were incubated for 6 h with 20 μg/ml Dil-oxLDL (Thermo Scientific) at 37°C, washed twice with PBS and resuspended in 400 μl PBS + 2% FCS+ 1 mM EDTA for immediate FACS analysis.
To measure the incorporation Dil-labeled oxidized LDL (oxLDL) by M1- and M2a-like macrophages, cells (1–2 × 105) were cultured for 24 h in HBSS supplemented with 0.3% bovine serum albumin in the presence or absence of 1000 ng/ml NETs. Next, cells were incubated for 6 h with 20 μg/ml Dil-oxLDL (Thermo Scientific) at 37°C, washed twice with PBS and resuspended in 400 μl PBS + 2% FCS+ 1 mM EDTA for immediate FACS analysis.
3
Characterization of Macrophage Polarization
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The spleen, peritoneal macrophages (PMs), and polarized peripheral blood mononuclear cells (pPBMC) were harvested and lysed with red blood cell lysing buffer (420,301; Biolegend). Cells were incubated with an Fc receptor blocker CD16/32 (eBioscience, San Diego, CA, USA) to reduce nonspecific antibody binding prior to adding staining antibodies. For live/dead cell separation 7-AAD (Invitrogen, Carlsbad, CA, USA) staining was used. Cells were labeled with APC anti-mouse F4/80, PerCP-Cyanine5.5 anti-mouse CD45, FITC anti-mouse CD11b, Brilliant Violet 421 anti-mouse CD206, Brilliant Violet 650 anti-mouse CD80 (clone BM8, clone 30-F11, clone M1/70, clone C068C2, and clone 16-10A1 respectively; Biolegend). For intracellular labeling, cells were stimulated with lipopolysaccharide (LPS) (10 μg/mL; Sigma–Aldrich, St. Louis, MO, USA), 3 μg/mL brefeldin A (Biolegend) for 5 h at 37 °C25 (link). The cells were stained with Brilliant Violet 605 anti-mouse IL-10, PE/Dazzle 594 tumor necrosis factor-alpha (TNF-α) (clone JES5-16E3 and clone 506,346, respectively; Biolegend), and PE anti-mouse inducible nitric oxide synthase (INOS) (clone CXNFT, respectively; eBioscience) and then analyzed using an CytoFLEX flow cytometer (Beckman Coulter, Miami, FL, USA). The data were analyzed by using FlowJo software (V.10.4, FlowJo, USA).
4
Targeted Delivery of Abemaciclib and IMD-0354
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Abemaciclib was purchased from MedChemExpress Co. Ltd. IMD‐0354 was purchased from Selleckchem Co. Ltd. MAL‐PEG2000‐NHS was purchased from Beijing Kaizheng Biotech Co. Ltd (Beijing, China). Poly (L‐lysine) (PLL, Mw = 3–7 w), 2,3‐Dimethylmaleic Anhydride (DMA), Cis‐aconitic acid anhydride (CA), 3,4,5,6‐Tetrahydrophthalic anhydride (TDA) and succinic anhydride (SA) were purchased from Sigma‐Aldrich Co. Ltd (American). NGR peptide (sequence: GCNGRCGC) was obtained from Shanghai Apeptide Co. Ltd (Shanghai, China). Polyamindoamine (PAMAM‐G5, Mw = 28 826) was purchased from CY dendrimer technology Co. Ltd (Weihai, China). Cell Cycle and Apoptosis Analysis Kit was the product of Beyotime Biotechnology Co. Ltd (Shanghai, China). Alexa Fluor 647 anti‐mouse F4/80, Brilliant Violet 421 anti‐mouse CD206, PerCP/Cy5.5 anti‐mouse F4/80, Alexa Fluor 488 anti‐mouse CD86, APC anti‐mouse CD206, APC anti‐mouse CD3, FITC anti‐mouse CD4, PE anti‐mouse CD8, PE anti‐mouse CD25, and Alexa Fluor 488 anti‐mouse Foxp3 were purchased from Biolegend. ELISA kits were obtained from Dakewei Co. Ltd (Nanjing, China). All other reagents and solvents were obtained from Sinopharm Co., Ltd (Shanghai, China) and Sigma‐Aldrich Co., Ltd (Shanghai, China).
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