Mononuclear cells were separated from the central nervous system (CNS) of mice using standard methods in the field, as described previously (29 (link)). Briefly, perfusion of the mice proceeded intracardially with PBS, and the whole brains of metastases models and brain tumors of stereotactic intracranial transplantation models were extracted and homogenized. Mononuclear cells were isolated by 25% discontinuous Percoll gradients (Sigma, St. Louis, MO, USA). The purified cells were stained with FITC anti-mouse CD3ε antibody, APC/Cy7 anti-mouse CD4 antibody, PE anti-mouse CD8a antibody, FITC anti-mouse/human CD11b antibody, PerCP anti-mouse CD45 antibody, PE anti-mouse CD86 antibody, PE/Cy7 anti-mouse CD206 (MMR) antibody, APC anti-mouse F4/80 antibody, and BV421-Zombie Violet Fixable Viability Kit (Biolegend). Fc receptors (FcR) were blocked with anti-mouse CD16/32. Antibody incubations were performed on ice, with the cells being fixed in 1% paraformaldehyde and analyzed on a BD LSRFortessa (BD Bioscience). The percentages of microglial cells and macrophages were obtained through counting the mean total number of mononuclear cells isolated from the CNS per mouse. Populations of CD11b+CD45low F4/80+-activated microglial cells and CD11b+CD45high F4/80+ peripheral macrophages were analyzed.
Apc anti mouse f4 80 antibody
Manufactured by BioLegend
Sourced in United States
The APC anti-mouse F4/80 antibody is a fluorescently-conjugated monoclonal antibody that specifically binds to the F4/80 antigen, which is expressed on the surface of mature mouse macrophages. This antibody can be used for the identification and characterization of mouse macrophages in flow cytometry applications.
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Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Fitc anti mouse human cd11b antibody, by BioLegend (2 mentions)
Pe anti mouse cd206 mmr antibody, by BioLegend (2 mentions)
Percoll gradient, by Merck Group (1 mentions)
Bv421 zombie violet fixable viability kit, by BioLegend (1 mentions)
Fvd520, by Thermo Fisher Scientific (1 mentions)
Anti apc microbeads, by Miltenyi Biotec (1 mentions)
Celltracker red cmptx dye, by Thermo Fisher Scientific (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Abcam (1 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Ab109497, by Abcam (1 mentions)
Ab90529, by Abcam (1 mentions)
Ab209845, by Abcam (1 mentions)
Fitc anti mouse cd45 antibody, by BioLegend (1 mentions)
Ag 25b 006 c100, by Adipogen (1 mentions)
Ag 20b 0042 c100, by Abcam (1 mentions)
Pe cyanine7 anti mouse cd86 antibody, by BioLegend (1 mentions)
Pe anti mouse human cd11b antibody, by BioLegend (1 mentions)
11 protocols using apc anti mouse f4 80 antibody
1
Isolation of CNS Immune Cells
2
Zymosan-Induced Peritoneal Macrophage Isolation
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In addition, we observed the above phenomena using confocal microscopy (TiEA1R, NIKON INSTECH Co. Ltd., Tokyo Japan) under the conditions of with or without AIM coating. Debris was stained with FVD520 (eBioscience). The peritoneal cells derived from the zymosan model mice were stained with APC anti-mouse F4/80 antibody (BioLegend, San Diego, CA) and incubated with Anti-APC microbeads (Miltenyi Biotec). F4/80 positive cells were separated using a MACS LS column (Miltenyi Biotec) stained with CellTracker™ Red CMPTX Dye (10 µM) (Thermo Fisher Scientific).
3
Cell Uptake Efficiency of Nanoparticles
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The cell uptake effciency of NPs were detected by CLSM and FCM. 1.5 mL of RAW 264.7 (200,000 cells/well) were seeded in dishes and incubated for 24 h (with or without 100 ng/mL LPS). Cells were washed three times with PBS, treated with 1.5 mL DMEM containing HAZ@FITC or ZIF@FITC (60 µg/mL) for specific time point. Then, medium was removed and cells were washed three times with PBS. For CLSM, cells were stained with DAPI under 37℃ for 15 min after fixation, images were immedately recorded. For FCM, after incubated with FITC-loaded NPs for 1 and 3 h, cell suspensions were centrifuged for 6 min at 1500 rpm at 4 °C. Supernatants were removed and RAW 264.7 pellets were stained with APC anti-mouse F4/80 antibody (Biolegend, USA), FITC and APC fluorescence of each well was detected by FCM.
4
Immunohistochemical Analysis of Mouse Tissue
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Mouse tissue sections were fixed in 4% paraformaldehyde for 15 min, washed in PBS and permeabilized with 0.1% Triton X-100 for 10 min. After blocked with 5% goat serum for 1 h at room temperature, they were incubated with primary antibodies at 4°C overnight. Then, samples were incubated with secondary antibodies and DAPI (1:1000) at room temperature for 1 h, and further imaged with a confocal laser scanning microscope (Olympus, Lake Success, LY). The following antibodies were used: mouse anti-occludin (1:200, #33-1500, Thermo Fisher), APC anti-mouse F4/80 antibody (1:50, #123115, BioLegend), Alexa Fluor 488-conjugated anti-mouse IgG (1:1000, ab150113, abcam).
5
Immunodetection of Inflammasome Components
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Rabbit monoclonal Anti-ASC (AG-25B-006-C100, AdipoGen, San Diego, CA, USA), mouse monoclonal anti-NLRP3 (AG-20B-0014-C100), rabbit monoclonal anti-caspase-1 (p20) (AG-20B-0042-C100), Rabbit monoclonal anti-GSDMD (ab209845, ABCAM, Cambridge, UK), rabbit monoclonal anti-PBR (ab109497, ABCAM), mouse Antiβ-actin (SJ190a9b68548, Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-IL-1β (12242, CST, Danvers, MA, USA), rabbit monoclonal anti-Iba1 (PA5-21274, Invitrogen), mouse monoclonal anti-Iba1 (019-19741, WAKO, Osaka Japan), anti-cytochrome C antibody (ab90529, Abcam) FITC anti-mouse CD45 antibody (103108, BioLegend, San Diego, CA, USA), APC anti-mouse F4/80 antibody (123116, BioLegend), PE/Cyanine7 anti-mouse CD86 antibody (105014, BioLegend), PE anti-mouse/human CD11b antibody (101208, BioLegend).
6
Isolation and Phenotyping of Immune Cells in EAE Mice
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The spleen lymphocytes of EAE mice (on the 20th day after immunization) were separated with Ficoll density gradient centrifugation. Peritoneal macrophages were isolated as previously described (7 (link)), washed twice with FACS (containing 2% fetal bovine serum), then stained with APC anti-mouse F4/80 antibody (BioLegend, 123116) and PE anti-mouse MHC-II antibody (BioLegend, B117132), incubated at 37°C for 30 min, and then washed once with FASC and resuspended in 1% paraformaldehyde. For staining of spleen cells, firstly, the cells were incubated with cell stimulation cocktail plus protein transport inhibitors (Invitrogen, 2260623) for 4–6 h, washed with FACS once for staining with PerCP anti-mouse CD4 antibody (BioLegend, 100538), and then washed with FACS; 200 μl fixation/permer buffer (Invitrogen, 2220750) was added at 4°C overnight, and then permer buffer dilution washing and staining were done on the APC anti-mouse IFN-γ antibody (BioLegend, 505810), PE anti-mouse IL-17 antibody (BioLegend, 506904), and APC anti-mouse Foxp3 antibody (eBioscience, E07303-1635), and finally washing with permer buffer and resuspension were performed.
7
LPS-Induced Macrophage Analysis in WT and NFIA-Tg Mice
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Normal chow-fed WT or NFIA-Tg mice were treated with either 0.3 mg/kg body weight LPS or saline for 12 h. FACS analysis of iWAT SVF was performed using APC anti-mouse F4/80 antibody (BioLegend, 123116) and FITC anti-mouse/human CD11b antibody (BioLegend, 101205). Flow cytometry and cell sorting were performed using the SH800 cell sorter (SONY).
8
Macrophage Subpopulation Analysis
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100 μL of suspension fluid was added to 0.25 μg APC anti-mouse F4/80 antibody (BioLegend), 1.0 μg PE anti-mouse CD86 antibody (BioLegend), and 0.125 μg FITC anti-mouse CD206 antibody (BioLegend), respectively. Homogenized solutions were placed in the dark at 4°C for 30 minutes, followed by addition of 200 μL FACS buffer, and centrifuged at 4°C, 300 ×g for 5 minutes. After removal of supernatant, the cellular solutions were washed with FACS buffer, followed by addition of 1 mL FACS buffer to resuspend cellular samples. Finally, cellular aggregates were broken up and analyzed with BD FACSCanto II, APC anti-mouse F4/80 antibody-specific M1 macrophage (F4/80+), APC anti-mouse F4/80 antibody, and FITC anti-mouse CD206 antibody-specific M2 macrophage (F4/80+, CD206+). FlowJo software was used to analyze the percentage of macrophages in the lung tissues and the proportions of M1 and M2 macrophages in the lung and tumor tissue samples.
Percentage of macrophages: macrophages (F4/80+)/cells.
Percentage of M1 macrophages: M1 (F4/80+, CD86+)/macrophages (F4/80+).
Percentage of M2 macrophages: M2 (F4/80+, CD206+)/macrophages (F4/80+).
Percentage of macrophages: macrophages (F4/80+)/cells.
Percentage of M1 macrophages: M1 (F4/80+, CD86+)/macrophages (F4/80+).
Percentage of M2 macrophages: M2 (F4/80+, CD206+)/macrophages (F4/80+).
9
Flow Cytometric Profiling of Renal Immune Cells
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Flow cytometry was performed as previously described (Zhao et al., 2018 (link)). Briefly, kidneys were weighed and minced. A collagenase solution (1 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) was used for renal digestion for 30 min at 37°C. A 100-μm cell strainer (Fisher Scientific) was used, together with a 1-ml syringe plunger, to acquire single cells. Cell pellets were washed and resuspended for further experiments. Anti-Mouse CD16/CD32 (553141; RRID:AB_394656 , clone 2.4G2; BD Biosciences, San Jose, CA, United States) was used for non-specific Fc block. Antibodies used in this assay were acquired from BioLegend, San Diego, CA, United States; they include BV421 anti-mouse CD45 (RRID:AB_2562559 ), APC anti-mouse F4/80 antibody (RRID:AB_893481 ), PE anti-mouse CD206 (MMR) antibody (RRID:AB_10895754 ), FITC anti-mouse CD86 antibody (RRID:AB_313149 ), PerCP/Cyanine5.5 anti-mouse/human CD11b (RRID:AB_893232 ), APC/Cyanine7 anti-mouse CD3 antibody (RRID:AB_2242784 ), FITC anti-mouse CD4 antibody (RRID:AB_312713 ), and APC anti-mouse CD8a antibody (RRID:AB_312750 ). Data were acquired on a FACS Calibur cytometer [Becton Dickinson (BD), Bedford, MA, United States] and analyzed using FlowJo software (Tree Star, Ashland, OR, United States).
10
Phenotypic Analysis of BV2 Microglia
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BV2 cells were filtered through a 70 μm cell filter and incubated with Zombie NIR™ Dye (Biolegend, USA) for 15 min, followed by termination with Cell Staining Buffer. FITC anti-rat/human CD11b antibody (Biolegend, USA), APC anti-mouse F4/80 Antibody (Biolegend, USA), and PerCP/Cyanine5.5 anti-mouse CD86 Antibody (Biolegend, USA) were then incubated for 40 min. The cells were permeabilized with Cyto-Fast™ Fix/Perm buffer for 20 min. Subsequently, the cells were incubated with PE anti-mouse CD206 (MMR) antibody (Biolegend, USA) in darkness at room temperature for 20 min and terminated with Cyto-Fast™ Perm/Wash buffer. Samples were collected using BD LSRFortessa flow cytometry (BD Bioscience, USA) and data analysis was performed using FlowJo.
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