Flow cytometric analysis of bone marrow, peripheral blood cells or enzymatically digested esophagus was conducted using the following antibodies: anti-CD11b (R&D), anti–GR-1 (BD Bioscience), anti–Siglec-F (BD Bioscience), anti-CCR3 (BD Bioscience), anti–PIR-A/B (ebioscience), IgG2b (ebioscience), anti-CD45 (ebioscience) and anti-CD11c (BD Bioscience). Cell counts were conducted using 123count beads (ebioscience) according to the manufacturers’ instructions. In all experiments, at least 50,000 events were acquired by (FACSCalibur, BD Bioscience), and data were analyzed using the Kaluza (BeckmanCoulter) or FlowJo (TreeStar) softwares.
Anti gr 1
Manufactured by BD
Sourced in United States
Anti-Gr-1 is a laboratory reagent used in flow cytometry applications. It is a monoclonal antibody that specifically binds to the Gr-1 antigen, which is expressed on the surface of certain immune cells. The primary function of Anti-Gr-1 is to facilitate the identification and characterization of Gr-1-positive cell populations within a sample.
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Lab products found in correlation
Anti cd11b, by BD (23 mentions)
Anti cd4, by BD (13 mentions)
Anti b220, by BD (13 mentions)
Anti cd3, by BD (11 mentions)
Anti cd11c, by BD (10 mentions)
Anti cd8, by BD (10 mentions)
Anti ter119, by BD (9 mentions)
Anti f4 80, by BD (6 mentions)
Anti nk1, by BD (6 mentions)
Anti mac 1, by BD (5 mentions)
Anti cd19, by BD (4 mentions)
Anti cd45, by BioLegend (4 mentions)
Anti cd3, by BioLegend (4 mentions)
Anti ly6g, by BD (4 mentions)
Anti cd8a, by BD (4 mentions)
Anti cd3e, by BD (4 mentions)
Anti cd69, by BD (3 mentions)
Anti c kit, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Anti cd45, by BD (3 mentions)
49 protocols using anti gr 1
1
Multiparameter Flow Cytometry of Immune Cells
2
Multiparametric Flow Cytometry Analysis of Immune Cell Subsets
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Surface and intracellular staining were performed as previously described [28 (link)]. Single immune cell populations from the spleen, lymph nodes or tumors were separated with a BD FACSAria II Cell Sorter. Flow cytometric analyses were performed with Flowjo (Tree Star). The following antibodies were used for cell staining: anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone DX5), anti-CD11b(clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD62L (MEL-14), anti-CD44 (clone IM7), anti-Foxp3 (clone FJK-16s), anti-Granzyme B (clone GB11), anti-CCR4 (clone 2G12), anti-CCR5 (clone HM-CCR5), anti-CXCR3 (clone CXCR3-173), NK1.1(clone PK136), anti-F4/80 (clone BM8), anti-Gr-1 (clone RB6-8C5), anti-interferon-γ (IFN-γ, clone XMG1.2), and anti-NK1.1 (clone PK136), CD4 blocking mAb (clone GK1.5), and CD8 blocking mAb (clone 53-6.7).
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)
3
Multiparametric Flow Cytometry Immunophenotyping
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Single-cell suspensions (1 × 106 cells) were incubated at 4 °C for 10 min with α-CD16/32 (clone 2.4G2, from BioExpress, Kaysville, UT, USA) to reduce non-specific binding. Cells were labeled with various combinations of fluorochrome-conjugated monoclonal antibodies (mAbs) and incubated at 4 °C for 25 min. The following mAbs were used: anti-TCRβ (H57-597), anti-CD8 (53–6.7), anti-CD49b (DX5), anti-IFNγ, anti-CD11b (M1/70), anti-NKG2D (CX5) from eBioscience™; anti-CD19 (1D3), anti-CD4 (RM4-5), anti-F4/80 (T45-2342), anti-NK1.1 (PK136), anti-Ki-67 (B56), anti-CD69 (H1-2F3), anti-Ly6C (AL-21), anti-Gr1 from BD Biosciences™, anti- Rae- Pan specific from Milteney biotech, anti-CD43 (1B11) activation- Glycoform from BioLegend™ and Live/Dead Fixable Yellow Dead Cell Stain from Invitrogen™. The intracellular staining of IFNγ and Granzyme B was performed using Cytofix/Cytoperm protocols (BD Biosciences™). Intracellular staining of Foxp3 was carried out using a Foxp3 staining kit (eBioscience™) according to the manufacturer’s protocol. Cells were acquired using BD LSRFortessa or Thermofisher Attune NxT flow cytometers, and data was analyzed using Kaluza 1.3 Analysis software (Beckman Coulter) or FlowJo (V10).
4
Isolation and Flow Cytometry of Neutrophils
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Flow cytometry analysis was performed on a LSRII or LSRFortessa (BD Biosciences). To isolate neutrophils from the feet, tissue was diced and incubated at 37°C for 45 min with shaking at 200 rpm (TissueLyserII, Qiagen), in DMEM containing 1 mg/mL Collagenase Type IV (Worthington Biochemical). Cells were then strained through a 70 μM strainer and washed twice in phosphate buffered saline (PBS). Neutrophils from bone marrow and feet were sorted on a FACSAria III (BD Biosciences). FcγR were blocked using TruStain FcX (BioLegend). Data were analyzed using FlowJo software (BD Biosciences). The following reagents were used: Zombie-NIR fixable viability kit (BioLegend), anti-B220 (eBioscience;RA36B2), anti-CD3 (WEHI;KT3.1.1), anti-CD4 (WEHI;GK1.5), anti-CD8 (WEHI;53.6.7), anti-CD11b (BioLegend;M1/70), anti-CD16/32 (WEHI;24G2), anti-CD34 (eBioscience;RAM34), anti-CD45.1 (BioLegend;A20), anti-CD45.2 (BioLegend;104), anti-CD48 (BD Bioscience;C2), anti-CD127 (eBioscience;A7R34), anti-CD135 (BioLegend;A2F10), anti-CD150 (BioLegend;TC15–12F12.2), anti-cKit (WEHI;ACK4), anti-Gr1 (WEHI;RB6–8C5), anti-Ly6G (BD;1A8), anti-Sca-1 (WEHI;Ly6A/E), anti-Siglec-F (Fisher;1RMN44N), AnnexinV-AF647 (BioLegend), and propidium iodide (Sigma).
5
Immunohistochemical Analysis of Kidney Inflammation
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Frozen sections of kidneys were stained with anti-Gr-1 (BD Pharmingen, USA) and anti-NLRP3 (CST, USA) followed by treatment with HRP-conjugated anti-rat IgG (Dako), and were visualized using diaminobenzidine (DAB) and hematoxylin as counterstaining. Frozen sections of kidneys were treated with anti-WT-1 (Merck Millipore, Bedford, MA, USA) followed by treatment with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA). MDSCs were collected and stained with anti-Nrf2 (Abcam, USA) followed by treatment with HRP-conjugated anti-rat IgG (Dako). Fluorescence images were captured by a laser scanning confocal microscope (FV3000, Olympus Corporation, Japan).
6
Isolation and Flow Cytometry of Neutrophils
7
Quantifying Gr1+ Cells and MPO Levels
8
Multicolor Flow Cytometry Analysis of Hematopoietic Stem and Progenitor Cells
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Numbers of hematopoietic cells derived from BM were analyzed by multicolor flow cytometry (BD Aria, BD Bioscience). These cell populations were further analyzed with FlowJo software for phenotypical identification. Hematopoietic progenitor cells (HPCs, Lin-Sca-1-c-Kit+), Lin-Sca-1+c-Kit+ (LSK) cells, and hematopoietic stem cells (HSCs, CD150+CD48-LSK) from BM were characterized with the following lineage markers: PE-Cy7-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11b, anti-Gr-1, and anti-TER-119 (BD Biosciences); FITC or PE-conjugated anti-Sca-1 (BD Biosciences); APC-conjugated anti-c-Kit (BD Biosciences); PerCP/Cy5.5-conjugated anti-CD150 (eBioscience); or APC-Cy7-conjugated anti-CD48 (BD Biosciences). SA-β-gal activity and mitochondrial superoxide anion levels were assessed using C12FDG (Molecular Probes) and MitoSoxTM Red (Invitrogen), respectively. Expression of p16INK4a was determined with Alexa Flour 488-conjugated antibody (Santa Cruz Biotechnology) after fixation and permeabilization (BD Bioscience).
9
Immunophenotyping of Tumor-Infiltrating Immune Cells
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Frozen sections of resected tumors were cut into 8‐μm serial slices. Slices were fixed in 4% paraformaldehyde for 5 min at room temperature, followed by serum‐free blocking protein (Dako). Samples were subsequently stained with primary antibodies, such as anti‐CD4 (1:50; BD Pharmingen), anti‐CD8 (1:150; BD Pharmingen), anti‐PD‐1 (1:100; Abcam), anti‐Foxp3 (1:400; Novus Biologicals) and anti‐dendritic cell marker which reacts with dendritic cell inhibitory receptor 2 (1:50; Novus Biologicals), and anti‐Gr‐1 (1:100; BD Pharmingen) antibodies overnight at 4°C. After washing, fluorescence‐labeled secondary antibodies (Alexa Fluor 594 goat anti‐rat, Alexa Fluor 488 goat anti‐rabbit and Alexa Fluor 488 donkey anti‐goat IgG H + L [1:250, Life Technologies]) were then applied to the sections for 30 min at room temperature. DAPI (Vector Laboratory) was used for nuclear staining. Stained slides were finally imaged using an Olympus BX61 scanning fluorescence microscope. For quantification data, counting was performed in three random fields at ×200 magnification per tumor tissue specimen.
10
Immunohistochemical Analysis of Kidney
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Frozen sections of kidneys were stained with anti-CD11b (BD Pharmingen, USA) or anti-Gr-1 (BD Pharmingen, USA) followed by treatment with horseradish peroxidase-conjugated anti-rat IgG (Dako), and were visualized using diaminobenzidine (DAB) and hematoxylin as counterstaining. Frozen sections of kidneys were treated with anti-Wilms’ tumor protein (WT-1) (Merck Millipore, Bedford, MA, USA) or anti-Nephrin (Merck Millipore, Bedford, MA, USA), followed by treated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Sections were analyzed by laser scanning confocal microscope (FV3000, Olympus Corporation, Japan).
To investigate oxidative stress in the kidney tissues, DHE immunofluorescence staining was performed. Frozen, optimal cutting temperature-embedded kidney tissue was cryosectioned into 10-μm-thick sections, which were stained with 10 µmol/l dihydroethidium (DHE, Molecular Probes) solution (Invitrogen, Carlsbad, CA, USA). Images were obtained by laser scanning confocal microscope (FV3000, Olympus Corporation, Japan).
To investigate oxidative stress in the kidney tissues, DHE immunofluorescence staining was performed. Frozen, optimal cutting temperature-embedded kidney tissue was cryosectioned into 10-μm-thick sections, which were stained with 10 µmol/l dihydroethidium (DHE, Molecular Probes) solution (Invitrogen, Carlsbad, CA, USA). Images were obtained by laser scanning confocal microscope (FV3000, Olympus Corporation, Japan).
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