Inducible nitric oxide synthase (iNOS, M1 marker) and arginase-1 (Arg-1, M2 marker) were used as markers to evaluate the polarization state of macrophages cultured on various specimens. After being fixed by 4% paraformaldehyde (PFA) for 20 min, permeabilized in 0.3% Triton-X for 10 min and blocked with 10% bovine serum albumin (BSA) solution for 2 h, the cells were treated with iNOS (Abcam, USA) and Arg-1 (Abcam, USA) primary antibody following the manufacturer’s instructions. Thereafter, the as-treated cells were incubated with Alexa Fluor 594 and 488 conjugated secondary antibody (Abcam, USA) for iNOS and Arg-1, respectively. Subsequently, the specimens were stained by 4′-6-diamidino-2-phenylindole (DAPI; Sigma, USA) for cell nuclei before the observation using a confocal laser microsope (Nikon, Japan).
Arg 1
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Arg-1 is an enzyme that catalyzes the conversion of L-arginine to L-ornithine and urea. It plays a key role in the urea cycle, which is the primary pathway for the disposal of nitrogen in many organisms.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (6 mentions)
β actin, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
β actin, by Proteintech (3 mentions)
Pd l1, by Abcam (3 mentions)
Cd206, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
Tsg101, by Abcam (3 mentions)
Iba 1, by Abcam (3 mentions)
Cd11b, by Abcam (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Alexa fluor 594, by Abcam (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Bcl 2, by Abcam (2 mentions)
46 protocols using arg 1
1
Macrophage Polarization Evaluation Protocol
2
Macrophage Phenotype Evaluation Protocol
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For cell surface examination, macrophages were incubated in 1% bovine serum albumen (BSA)-PBS buffer at room temperature for 20 min. Thereafter, cells were incubated in the M1 antibody marker CD68 or the M2 antibody marker CD200R (eBioscience, San Diego, CA, USA) in a dark room at 4°C.
For intracellular staining, cells were stimulated with 1 μg/ml brefeldin A (Sigma, USA) for 6 h at 37°C under 5% CO2/95% air atmosphere, and stained for 20 min at room temperature in 0.3% saponin/1% BSA-PBS buffer. After rinsing, cells were incubated for 1 h at 4°C with the following antibodies: iNOS (eBioscience lnc.); arginase 1 (Arg-1) (eBioscience lnc.); Nrf2 (eBioscience lnc.). Expression of iNOS and Arg-1 are key markers associated with M1 and M2 phenotypes, respectively.6 (link)For measurement of T helper 1 (Th1) and regulatory T cells (Treg) cells, anti-mouse CD4 FITC (Abcam) was added and cells were incubated for 30 min at 4°C. Thereafter, cells were incubated in anti-mouse Foxp3 PE (Abcam) and incubated for 30 min at 4°C.
Subsequently, all cells were subjected to flow cytometry detection (Becton Dickinson Biosciences, San Jose, CA, USA) and analysed using CellQuest software.
For intracellular staining, cells were stimulated with 1 μg/ml brefeldin A (Sigma, USA) for 6 h at 37°C under 5% CO2/95% air atmosphere, and stained for 20 min at room temperature in 0.3% saponin/1% BSA-PBS buffer. After rinsing, cells were incubated for 1 h at 4°C with the following antibodies: iNOS (eBioscience lnc.); arginase 1 (Arg-1) (eBioscience lnc.); Nrf2 (eBioscience lnc.). Expression of iNOS and Arg-1 are key markers associated with M1 and M2 phenotypes, respectively.6 (link)For measurement of T helper 1 (Th1) and regulatory T cells (Treg) cells, anti-mouse CD4 FITC (Abcam) was added and cells were incubated for 30 min at 4°C. Thereafter, cells were incubated in anti-mouse Foxp3 PE (Abcam) and incubated for 30 min at 4°C.
Subsequently, all cells were subjected to flow cytometry detection (Becton Dickinson Biosciences, San Jose, CA, USA) and analysed using CellQuest software.
3
Western Blot Analysis of Arg-1 and iNOS
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Samples were subjected to 10% SDS–PAGE, transferred to a polyvinylidene fluoride membrane, blocked with blocking buffer (Tris-buffered saline Tween-20 [TBST], containing 5% nonfat dry milk) for one hour at room temperature and incubated with primary antibodies against Arg-1 (1:1000; Abcam, Cambridge, MA, USA), iNOS-1 (1:1000; Stem Cell Technology), and GAPDH (1:1000, Cell Signaling Technology, Beverly, MA, USA) overnight. The membranes were then washed three times with TBST and probed with horseradish peroxidase-conjugated secondary antibodies (1:2000, Proteintech) for one hour at room temperature. The membranes were then washed with TBST and exposed to a molecular imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK) [20 (link)].
4
Western Blot Analysis of Arg-1, iNOS, and β-actin
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5
Cellular Immunofluorescence Staining Protocol
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For cellular immunofluorescence staining, nanofiber membranes from each group that had been seeded with cells were placed in tissue culture plates, washed twice with PBS and then fixed with 4% paraformaldehyde for 15–20 min. Then, 0.5% Triton X-100 was added for 30 min of incubation. Bovine serum albumin (BSA) (3%) was added to the cells, which were incubated for 30 min for blocking. Subsequently, a primary antibody was added for incubation overnight at 4 °C. Then, the nanofiber membranes were washed three times with PBS and incubated with a fluorescently coupled secondary antibody for 1 h. DAPI staining solution was added dropwise, and incubation was continued for 5 min. Fluorescence confocal microscopy was used to observe and photograph the samples after they were washed with PBS. Three replicates were performed for each group, and three images of the field of view were acquired for each sample. Image J was used to obtain the average relative fluorescence intensity, by adjusting the threshold and confirming a suitable algorithm. Primary antibodies against iNOS (Abcam), Arg-1 (Abcam), and ASC (Novus) were used. Dilution of the antibodies are listed in Supplementary Table S6. The antibodies were diluted with the antibody dilution containing 3%BSA.
6
Quantifying Lung mRNA and Protein Levels
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Total RNA from the lung samples was isolated using TRIzol reagent (Takara Bio, Kusatsu, Japan) according to manufacturer’s instructions. Reverse transcription master mix (G490) and EvaGreen qPCR master mix (MasterMix-ER) were purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). ARG1, MRC1, Retnla, CHIL3, TNF, and IFNG mRNA levels were determined by quantitative reverse transcription (qRT)-PCR using GAPDH as a reference gene (Table 3 ). Expression levels of ARG1, CHIA, and Retnla (Abcam, Cambridge, United Kingdom) in lung tissue were also measured by western blotting using β-actin as loading control. Protein samples from the lungs were fractionated via SDS-PAGE. Three independent experiments were performed.
7
Western Blot Analysis of Macrophage Markers
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Cells or exosomes were collected and lysed in Laemmli buffer (Sigma, USA). The protein concentration was determined with BCA Protein Assay kit (Beyotime). Protein lysates were separated by SDS-PAGE and transferred onto NC filter membrane (Millipore). Then the NC filter membrane was subsequently incubated with the primary antibody: iNOS (Abcam), Arg1 (Abcam), p-STAT1 (Abcam), p-STAT6 (Abcam), STAT1 (Abcam), STAT6 (Abcam), SOCS2 (Abcam), PKM2 (Cell Signaling Technology, CST, Danvers, MA, USA), HIF-1α (Invitrogen, Life Technologies, Carlsbad, CA, USA), β-actin (Abcam). β-actin was used as a control. After incubation with secondary antibodies and ECL (enhanced chemiluminescence, Millipore), the protein bands were captured by Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Quantitative analysis of protein bands was conducted with ImageJ software.
8
Immunofluorescence Staining of M1/M2 Macrophages
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Immunofluorescence staining was performed to assess the activation of the C-C chemokine receptor type 7 (CCR7, M1 phenotype) and arginase-1 (Arg-1, M2 phenotype). After culturing for 4 days, the cells were fixed in paraformaldehyde (4%), then blocked with 1% bovine serum albumin (BSA) for 30 min and cultured with CCR7 (1:100, Abcam) and Arg-1 (1:100, Abcam) antibodies overnight at 4°C. The next day, the cells were rinsed with PBS, treated with the secondary antibodies, donkey anti-mouse Alexa Fluor 594 (1:200, Abcam) and donkey anti-rabbit Alexa Fluor 488 (1:200, Abcam), and incubated for 1 h in the dark. Then, 4ʹ,6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei for 10 min.
9
Tamoxifen-mediated Immunomodulation Assay
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Tamoxifen (non-pharmaceutical grade) was purchased from Sigma-Aldrich (St Louis, MO). Giemsa cytological stain was purchased from Sigma-Aldrich. Antibodies used for in situ hybridization, immunohistochemical and flow cytometric analysis were: tgfb1 (Cat # 407751, Advanced Cell Diagnostics, ACD); RFP (Cat # ab62341; 1:1000, Abcam, Cambridge, MA); Arg1 (Cat # ab91279; 1:1500, Abcam); iNOS (Cat # ab15323; 1:200, Abcam); CD64 (Cat # bs-3511R; 1:250, Bioss Antibodies, Woburn, MA); CCR2 (Cat # EPR20844; 1:400, Abcam); phospho-SMAD2/3 (Cat # PA5-37636; 1.500, Invitrogen CX3CR1 (Cat # bs-1728R; Bioss Antibodies); CD64 (Cat # bs-3511R; Bioss Antibodies); CD125/IL-5RA (Cat # bs-2601R, Bioss Antibodies); C1q (Cat # A0136; 1:500, Dako/Agilent Technologies, Santa Clara, CA); CD16/32 (clone 93; eBiosciences, San Diego, CA), CD11b (clone # M1/70; eFluo450, eBiosciences); Fixable Viability dye (Cat # 65-0865-14; eFluo780, eBiosciences); SigF (clone S17007L; PE-CF594, BD Biosciences, San Jose, CA); CD45 (clone 30-F11; PerCP5.5, Biolegend, San Diego, CA); CD11c (clone # N418; BV705, Biolegend); Ly6G (clone # 1A8; AF700, Biolegend); Ly6C (clone HK1.4, BV510, Biolegend); CD64 (clone X54-5/7.1; PE/Cy7, Biolegend); CD43 (clone # S11; PE, Biolegend); CD3 (clone # 17A2; BUV395, Biolegend). All other reagents were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA), or Sigma-Aldrich.
10
Protein Expression Analysis in Cells
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The cells were lysed in RIPA lysis buffer (Beyotime, China, P0013B) supplemented with protease inhibitors PMSF (Beyotime, China, ST506). Protein concentration were determined by using BCA protein assay kit (Beyotime, China, P0012S). The primary antibodies included iNOS (Abcam, ab178945, 1:1000), Arg1 (Abcam, ab233548, 1:2000), PGRN (Abcam, ab187070, 1:1000), PD-L1 (Abcam, ab213480, 1:1000), STAT3 (Cell Signaling Technology, 9139 T, 1:1000), pSTAT3 (Cell Signaling Technology, 9145 T, 1:1000), AKT (Cell Signaling Technology, 4685S, 1:1000), pAKT (Cell Signaling Technology, 4060 T, 1:1000), ERK1/2 (Cell Signaling Technology, 9102S, 1:1000), pERK1/2 (Cell Signaling Technology, 4377 T, 1:1000), and β-actin (proteintech, 66,009–1-Ig, 1:2000).
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