Renal tissues were ground in a strainer. After red blood cell lysis and washing with phosphate-buffered saline (PBS) 3 times, cells were marked using an anti-mouse-F4/80-PE (eBioscience, Shanghai, China) antibody. All samples were washed 3 times after the antibodies staining, and then subjected to processing with a fluorescence-activated cell sorter (FACS) LSR II (BD Bioscience, USA), and further analyzed by FlowJo software (Tomy Digital Biology Co., Ltd., Japan). At least 3 duplicates were measured for each group.
Anti mouse f4 80 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse F4/80 PE is a fluorochrome-conjugated antibody that binds specifically to the F4/80 antigen expressed on the surface of mouse macrophages. It is a useful tool for the identification and enumeration of mouse macrophages in flow cytometric analysis.
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Lab products found in correlation
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Facscanto 2 flow cytometer, by BD (1 mentions)
Apc cy7 anti mouse cd11b, by BD (1 mentions)
Apc anti mouse cd11c, by BioLegend (1 mentions)
Lung dissociation kit, by Miltenyi Biotec (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Anti mouse cd80 apc, by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Fc block, by BD (1 mentions)
Anti mouse rat foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti ly6g gr1 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd3e pe cy7, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest version 3, by BD (1 mentions)
Anti cd45r b220 pe cy7, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Liankebio (1 mentions)
Anti human cd86 pe, by Thermo Fisher Scientific (1 mentions)
15 protocols using anti mouse f4 80 pe
1
Isolation and Analysis of Renal Macrophages
2
Lung Cell Populations Analysis After SiO2-FITC Exposure
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On day 53, single cell suspensions were generated from lungs of NS and S mice exposed to SiO2-FITC or to corresponding SUP (n=5–7), dissociated with a lung dissociation kit (kit number 130-065-927; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and analyzed by flow cytometry, monitoring the FITC conjugate. Untreated mice were used as negative controls. To investigate the interaction of SiO2-FITC with different lung cell populations, cells were stained and gated as described previously.24 anti-mouse CD45 PE-Cyanine7, 7AAD, and anti-mouse F4/80 PE were obtained from eBioscience (San Diego, CA, USA), anti-mouse CD11b APC-Cy™7 from BD Biosciences (San Jose, CA, USA), anti-mouse CD11c APC from BioLegend (San Diego, CA, USA), and GR1 (LY6C/G) Pacific Orange™ from Invitrogen (Life Technologies, Carlsbad, CA, USA). Samples were measured in a BD FACSCanto II flow cytometer and data were analyzed using a FlowJo graphics application V9.7.2 (TreeStar Inc., Ashland, OR, USA).
Interstitial macrophages were defined as CD45+, Gr-1Low, F4/80+, CD11b+ but CD11c−; alveolar macrophages as CD45+, Gr-1Low, F4/80+, CD11c+ but CD11b−; neutrophils as CD45+, Gr-1High, and CD11b+; and lymphocytes as CD45+, but CD11b− and SSCLow.
Interstitial macrophages were defined as CD45+, Gr-1Low, F4/80+, CD11b+ but CD11c−; alveolar macrophages as CD45+, Gr-1Low, F4/80+, CD11c+ but CD11b−; neutrophils as CD45+, Gr-1High, and CD11b+; and lymphocytes as CD45+, but CD11b− and SSCLow.
3
Macrophage Purity and Apoptosis Assay
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To confirm the purity of macrophages, the cells isolated from the peritoneal fluid of the mice were incubated with anti-mouse F4/80-PE (eBioscience, USA) fluorescent antibodies for 30 min and were washed twice with staining buffer before measurement with a BD FACS Calibur flow cytometer (BD Biosciences, East Rutherford, NJ, USA). In brief, cells were captured via high forward scatter and high side scatter. Favorite cells were gated as shown in Region 1 (R1), of which PE-positive cells were selected. Results were expressed in the percentage of positive cells. The experiments were performed in triplicate.
To measure apoptosis, the cells treated with ox-LDL were washed with cold PBS and resuspended in 1 mL of 1× binding buffer, and then 100 μL of cells (1 × 105) was placed into individually labeled tubes. Annexin V-FITC was added to the cellular suspension, as per the manufacturer’s instructions, and a fluorescent sample of 10,000 cells was immediately analyzed with a BD FACS Calibur flow cytometer.
To measure apoptosis, the cells treated with ox-LDL were washed with cold PBS and resuspended in 1 mL of 1× binding buffer, and then 100 μL of cells (1 × 105) was placed into individually labeled tubes. Annexin V-FITC was added to the cellular suspension, as per the manufacturer’s instructions, and a fluorescent sample of 10,000 cells was immediately analyzed with a BD FACS Calibur flow cytometer.
4
Flow Cytometry Analysis of Macrophages
5
Multiparameter Flow Cytometry Analysis
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Isolated cells were incubated in 2% FBS and Fc block (BD Bioscience) for 20 min at 4°C and then stained with fluorescent-conjugated antibody. Most Abs used for flow cytometry analysis were purchased from BD Biosciences except for anti-mouse F4/80-PE (eBioscience), anti-mouse/rat Foxp3-APC (eBioscience), and anti-mouse CD317(BST2, PDCA-1)-PE (eBioscience). Data were obtained using FACSCanto (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR). For intracellular staining for IL-10, the cells were stimulated with PMA/ionomycin or anti-CD3 antibody and then fixing and permeabilization buffer were used as per manufacturer instructions.
6
Flow Cytometric Analysis of Cellular Markers
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Suspended cells from FLs were stained for FACS with the following antibodies: anti-mouse F4/80-PE (eBiosciences #12-4801-80), anti-Adra2b (Alomone Labs #AAR-021), anti-adducinβ (Santa Cruz # sc-376063), and anti-spectrinβ1 (Santa Cruz # sc-374309). For anti-Adra2b staining, we used an Alexa 647 conjugated Donkey anti-rabbit secondary antibody (Life Technologies). For adducinβ and spectrinβ1 staining, primary unconjugated antibodies were conjugated to AlexaFluor 647 using a primary antibody conjugation kit (Invitrogen # Z11235). Flow cytometry data was analyzed by FCS Express software, and gates were drawn based on unstained and single-color compensation controls from the same samples, using the same dyes and within the same experiment.
7
Differential Cell Analysis of Mouse BALF
8
Flow Cytometry Analysis of RAW Macrophages
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RAW macrophages were cultured in either RAW media described above or in conditioned media (CM) from MSCs for 24 hours. Following 24 hours 5 × 105 of RAW cells were washed twice with PBS containing 1% goat serum and resuspended with 10 μL of anti-mouse CD206 Alexa Fluor 647, Arginase 1 PE (AbD Serotec), and 40 μL of the 1% serum PBS buffer. For tumor samples collected in vivo, cells were digested and washed in PBS containing 1% goat serum and resuspended in 10 μL of anti-mouse F4/80 PE (Ebioscience) and CD206. For both assays, samples were incubated for 20 minutes on ice, washed with 1% serum PBS buffer, and analyzed with the FACSCalibur. Cells with an isotype control for each antibody were used to set up the gate for analysis. The data were analyzed by the FlowJo software (version 8.8.6; TreeStar, Inc.).
9
Immunophenotyping of Macrophage Activation
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AS-IV powder (C41H68O14, molecular weight: 784.9702, purity > 98%) was purchased from Shanghai Ronghe Co. (Shanghai, China). The following antibodies were purchased from eBiosciences (CA, USA): anti-human CD206 PE, anti-human CD86 PE, anti-human CD14 PerCP/Cy5.5, anti-mouse CD45 v450, anti-mouse CD11b FITC, anti-mouse F4/80 PE, anti-mouse CD206 Alexa-647, and anti-mouse iNOS APC. Antibodies against total and phosphorylated AMPKα, arginase 1 (Arg-1), CD31, and vascular endothelial growth factor A (VEGFA) were purchased from Cell Signaling Technology (MA, USA). IL4, IL13, IFN-γ, and LPS were purchased from Peprotech (NJ, USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Liankebio (Hangzhou, China). JetPrime transfection agent was obtained from Polyplus (NY, USA).
10
Purification and Phenotyping of Murine Peritoneal Macrophages
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We plated peritoneal macrophages on untreated Petri dishes and allowed them to adhere overnight. The next morning, we lifted cells by incubating for 5 min in ice-cold Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific) without calcium or magnesium and scraping gently. We next filtered cells through a 40 μm cell strainer, washed them with DPBS, and resuspended them in DPBS containing 5% FBS, 10 mM HEPES, 1 mM EDTA, and TruStain fcX™ antibody (BioLegend, San Diego, CA). We stained 105 to 106 cells with APC anti-mouse CD36 (clone: HM36; BioLegend), PE/Cy7 anti-mouse CD64 (clone: ×54–5/7.1; BioLegend), and PE anti-mouse F4/80 (clone: BM8; eBioscience) antibodies for 20 min on ice. We then washed and resuspended cells in DPBS and acquired data on a BD X-20 or BD LSR II flow cytometer (BD Biosciences, San Jose, CA). We analyzed and visualized data with FlowJo v10.
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