clone 1a8, by BioLegend - Product details

1

Flow Cytometry and Immunohistochemistry Antibody Sources

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Sources of the antibodies for flow cytometry analysis are listed as follow: mouse Fc Block (1:100; 553142; BD Biosciences), mouse CD45 (1:100; clone 30-F11; Biolegend), mouse Gr1 (1:100; clone RB6-8C5; Biolegend), mouse CD11b (1:100; clone M1/70; eBiosciences), mouse CD11c (1:100; clone N418; Biolegend), mouse F4/80 (1:100; clone MB8; Biolegend), mouse Ly6G (1:100; clone 1A8; Biolegend), mouse Ly6C (1:100; clone HK1.4; Biolegend), human ENTPD2 (1:25; PA5-26333; Sigma-Aldrich), mouse Entpd2 (1:25; ab150503; Abcam), human ENTPD1 (1:100; clone A1; Biolegend), and mouse Entpd1 (1:100; clone Duha59; Biolegend). Sources of the antibodies for immunohistochemistry are listed as follow: human ENTPD2 (1:200; ab150503; Abcam), human GLUT1 (1:1000; ab15309Abcam), and human CA9 (1:500; ab1508L; Abcam).

Chiu D.K., Tse A.P., Xu I.M., Di Cui J., Lai R.K., Li L.L., Koh H.Y., Tsang F.H., Wei L.L., Wong C.M., Ng I.O, & Wong C.C. (2017). Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma. Nature Communications, 8, 517.

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2

Tumor-Infiltrating Lymphocyte Analysis

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The significant differences in tumor volume between IDO-APT treated group and Scr-APT treated group began to appear Mice were euthanized 2 days after the third treatment and the tumor tissue was collected. Tumor tissues were cut into small pieces and digested with collagenase D and DNase I for 30 min at 37°C. After grinding and passing through the 200 meshes strainer, TILs were isolated from the dissociated cells by Ficoll-Hypaque (Dakewe Biotech). For intracellular cytokine analysis, cells were re-stimulated with 100 ng/mL PMA and 500 ng/mL ionomycin in the presence of protein transport inhibitor cocktail for 5 h. After staining with anti-CD45-PE-Cy7 and anti-CD8-FITC, cells were fixed and permeabilized and then stained with anti-TNF-α-PE or anti-IFN-γ-APC. For T_reg cells analysis, cells were stained with anti-CD45-PE-Cy7, anti-CD4-FITC, anti-CD25-PE, and anti-Foxp3-APC. For tumor-associated macrophages analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC (Cat 101205, Clone M1/70, Biolegend), and anti-F4/80-APC (Cat 123115, Clone BM8, Biolegend). For granulocytic or monocytic MDSC analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC, anti-Ly6C-PE (Cat 128007, Clone HK1.4, Biolegend) or anti-Ly6G-PerCP (Cat 127653, Clone 1A8, Biolegend). The stained cells were then measured by flow cytometry.

Zhu Z., Yang Z., Zhu C., Hu Z., Jiang Z., Gong J., Yuan Y., Chen X., Jin Y, & Yin Y. (2023). Development of a DNA aptamer targeting IDO1 with anti-tumor effects. iScience, 26(8), 107367.

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3

Adoptive Transfer of Tumor-Associated Monocytes

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Front legs, hind legs and hips were collected from female mTmG mice and bone marrow was flushed out. Bone marrow cells were incubated with Fc Block (1:50, CD16/CD32, BD Biosciences), stained with anti-Ly6G-APC (1:200, clone 1A8, Biolegend) and consequently negative selection for neutrophils was performed using magnetic columns (Miltenyi Biotec) as described previously61 (link). The Ly6G^- fraction was then stained with fluorochrome-conjugated antibodies (Supplementary Table 1). After gating out Lineage⁺ cells (CD3, CD8, CD4, NKp46, Ter119), Siglec F⁺, Sca1⁺ and cKIT⁺ cells, tdTomato⁺CD11b^intLy6G^-Ly6C⁺ monocytes were isolated with BD FACSARIA FUSION sorter with DIVA software (BD Biosciences). Between 1,5 and 2 x 10⁶ tdTomato⁺ monocytes were adoptively transfer into the tail vein of a tumor-bearing KEP mouse treated with control antibody or anti-CSF-1R. Antibody treatments started at a tumor size of 25mm² and one day after the second antibody injection (one week apart), monocytes were transferred. KEP mice were sacrificed four days later and tumors were isolated for flow cytometry analysis. Antibodies are listed in supplementary Table 1.

Salvagno C., Ciampricotti M., Tuit S., Hau C.S., van Weverwijk A., Coffelt S.B., Kersten K., Vrijland K., Kos K., Ulas T., Song J.Y., Ooi C.H., Rüttinger D., Cassier P.A., Jonkers J., Schultze J.L., Ries C.H, & de Visser K.E. (2019). Therapeutic targeting of macrophages enhances chemotherapy efficacy by unleashing type I interferon response. Nature cell biology, 21(4), 511-521.

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4

Macrophage Depletion in Mice for Immunological Studies

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One day before surgery, 200 μl of clodronate liposomes (5 mg/ml) or empty liposomes (Liposoma) was intravenously injected in C57BL/6 mice (10 to 12 weeks old). An additional 200 μl of clodronate liposomes or empty liposomes were intraperitoneally injected every 2 days until day 6. Mouse spleens were harvested and crushed, and red blood cells were lysed with red blood cell lysis buffer [ammonium chloride (8.3 g/liter) and 10 mM tris-HCl in distilled water]. Macrophage depletion was verified by resuspending splenocytes in TruStain FcX anti-CD16/32 (1 μg/ml; clone 93, BioLegend) antibodies to block nonspecific binding and 1:500 dilution of Zombie Aqua (BioLegend) diluted in PBS. Subsequently, splenocytes were labeled with the following antibodies: anti-CD11b PE (1 μg/ml; clone M1/70, BioLegend), anti-Ly6G BV421 (1 μg/ml; clone 1A8, BioLegend), and anti-F4/80 biotin (3 μg/ml; clone REA126, Miltenyi Biotech) conjugated to streptavidin APC/Fire 750 (0.4 μg/ml; BioLegend) diluted in flow cytometry buffer (PBS with 1% BSA and 5 mM EDTA). Samples were acquired on a BD FACS Fortessa X20 and analyzed with FlowJo software (TreeStar Inc.).

Julier Z., Karami R., Nayer B., Lu Y.Z., Park A.J., Maruyama K., Kuhn G.A., Müller R., Akira S, & Martino M.M. (2020). Enhancing the regenerative effectiveness of growth factors by local inhibition of interleukin-1 receptor signaling. Science Advances, 6(24), eaba7602.

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5

Adoptive Transfer of Tumor-Associated Monocytes

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Front legs, hind legs and hips were collected from female mTmG mice and bone marrow was flushed out. Bone marrow cells were incubated with Fc Block (1:50, CD16/CD32, BD Biosciences), stained with anti-Ly6G-APC (1:200, clone 1A8, Biolegend) and consequently negative selection for neutrophils was performed using magnetic columns (Miltenyi Biotec) as described previously61 (link). The Ly6G^- fraction was then stained with fluorochrome-conjugated antibodies (Supplementary Table 1). After gating out Lineage⁺ cells (CD3, CD8, CD4, NKp46, Ter119), Siglec F⁺, Sca1⁺ and cKIT⁺ cells, tdTomato⁺CD11b^intLy6G^-Ly6C⁺ monocytes were isolated with BD FACSARIA FUSION sorter with DIVA software (BD Biosciences). Between 1,5 and 2 x 10⁶ tdTomato⁺ monocytes were adoptively transfer into the tail vein of a tumor-bearing KEP mouse treated with control antibody or anti-CSF-1R. Antibody treatments started at a tumor size of 25mm² and one day after the second antibody injection (one week apart), monocytes were transferred. KEP mice were sacrificed four days later and tumors were isolated for flow cytometry analysis. Antibodies are listed in supplementary Table 1.

Salvagno C., Ciampricotti M., Tuit S., Hau C.S., van Weverwijk A., Coffelt S.B., Kersten K., Vrijland K., Kos K., Ulas T., Song J.Y., Ooi C.H., Rüttinger D., Cassier P.A., Jonkers J., Schultze J.L., Ries C.H, & de Visser K.E. (2019). Therapeutic targeting of macrophages enhances chemotherapy efficacy by unleashing type I interferon response. Nature cell biology, 21(4), 511-521.

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6

Multicolor Flow Cytometry Profiling of Hematopoietic Cells

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Isolated cells were stained at 4°C in FACS buffer (PBS supplemented with 2.5% bovine serum albumin) with and hematopoietic lineage markers including Ly6G (BioLegend, clone 1A8, 1:600), CD11b (BioLegend, clone M1/70, 1:600) and Ter119 (BioLegend, clone TER-119, 1:600). This was followed by a second staining for NK1.1 (BioLegend, clone PK 136, 1:600), Thy1(CD90.2, BioLegend, clone 53-2.1 1:600), and Ly6C (BioLegend, clone HK1.4 1:600). Cell suspensions were labeled with DAPI just prior to flow cytometric analysis to allow exclusion of dead cells. Doublets, erythrocytes, and dead cells were excluded by forward scatter, Dapi, and Ter119. Neutrophils were identified as (Ter119^low/CD11b^high/Ly6G^high). Monocytes were identified as (Ly6G^low/Ter119^low/CD11b^high/Ly6C^high). NK or T cells were identified as (CD11b^low/Ly6G^low/(Nk1.1^high or CD90.2^high)) respectively. The Cre-induced fraction of each hematopoietic lineage subset was determined based on the fraction that was tdTomato^high. Data was acquired by Sony sorter MA900 at UCSD and analyzed with FlowJo software.

Taghdiri N., Calcagno D.M., Fu Z., Huang K., Kohler R.H., Weissleder R., Coleman T.P, & King K.R. (2021). Macrophage calcium reporter mice reveal immune cell communication in vitro and in vivo. Cell Reports Methods, 1(8), 100132.

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7

BAL Cell Subset Identification

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The BAL cell populations were defined as follows: alveolar macrophages (high autofluorescence, CD11c⁺, CD11b^−/low, Ly6G⁻), plasmacytoid dendritic cells (pDCs; CD11c^+/int, mPDCA⁺, low autofluorescence), and neutrophils (CD11c⁻, CD11b⁺, Ly6G⁺). The following antibodies were used to determine marker positivity: PE‐conjugated anti‐mouse Ly6G (0.25 µg, clone 1A8, Biolegend), APC‐conjugated anti‐mouse mPDCA (1 µg, clone JF05‐1C2.4, Miltenyi Biotec), PE‐Cy‐7‐conjugated anti‐mouse‐CD11c (0.06 µg clone HL3, BD Biosciences), and Alexa Fluor 700‐conjugated anti‐mouse CD11b (0.25 µg, Clone M1/70, Thermo Fisher).

Elkhatib S.K., Alley J., Jepsen M., Smeins L., Barnes A., Naik S., Ackermann M.R., Verhoeven D, & Kohut M.L. (2021). Exercise duration modulates upper and lower respiratory fluid cellularity, antiviral activity, and lung gene expression. Physiological Reports, 9(20), e15075.

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8

Intravital Microscopy of Leukocyte-Endothelial Interactions

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Mice were placed in supine position and the right jugular vein was cannulated with a catheter for antibody injection. Intravital microscopy was performed after injection of a phycoerythrin (PE)‐conjugated antibody to Ly6G (1 µg, clone 1A8, 127608, BioLegend), an fluorescein (FITC)‐conjugated antibody to Ly6C (1 µg, HK1.1, 128022, BioLegend), and a PE‐conjugated antibody CD11b (1 µg, clone M1/70, 101208, BioLegend). Leukocyte‐endothelial interactions along the carotid artery were visualized using an Olympus BX51 microscope equipped with a Hamamatsu 9100‐02 electron multiplying CCD camera, and a 10 × saline‐immersion objective. Movies of 30 s were acquired and analyzed offline. One video per mouse was analyzed and video analysis was done in a blinded fashion.

Bozoglu T., Lee S., Ziegler T., Jurisch V., Maas S., Baehr A., Hinkel R., Hoenig A., Hariharan A., Kim C.I., Decker S., Sami H., Koppara T., Oellinger R., Müller O.J., Frank D., Megens R., Nelson P., Weber C., Schnieke A., Sperandio M., Santamaria G., Rad R., Moretti A., Laugwitz K., Soehnlein O., Ogris M, & Kupatt C. (2022). Endothelial Retargeting of AAV9 In Vivo. Advanced Science, 9(7), 2103867.

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9

Flow Cytometric Analysis of Immune Cells

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The primary antibodies used for flow cytometry were as follows: anti‐F4/80 (clone BM8, BioLegend), anti‐CD11c (clone N418, BioLegend), anti‐MHC II (clone 10‐3.6, BioLegend), anti‐PDCA1 (clone 927, BioLegend), anti‐Gr1 (clone RB6‐8C5, BioLegend), anti‐CD4 (clone GK1.5, BioLegend), anti‐CD8a (clone 53‐6.7, BioLegend), anti‐IFN‐γ (clone XMG1.2, BioLegend), anti‐CD44 (clone IM7, BioLegend), anti‐CD80 (clone 16‐10A1, BioLegend), anti‐CD62L (clone MEL‐14, BioLegend), anti‐CD11b (clone M1/70, BioLegend), anti‐CD86 (clone GL‐1, BioLegend), anti‐I‐A/I‐E (clone M5/114.15.2, BioLegend), DC Marker (clone 33D1, BioLegend), anti‐Siglec‐H (clone 551, BioLegend), anti‐CD3ε (clone 145‐2C11, BioLegend), anti‐CD19 (clone 6D5, BioLegend), anti‐Ly‐6G (clone 1A8, BioLegend) and anti‐CD335 (clone 29A1.4, BioLegend). Mononuclear cells were isolated from the spleen and draining lymph nodes. A total of 1 × 10⁶ cells were incubated with 1·5 mg mL⁻¹ antibody for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) for 30 min at 4°C. The cells were then washed and stained for 30 min at 4°C with antibodies diluted with permeabilization buffer. The cell suspensions were analysed on an LSRII flow cytometer (BD Biosciences), and the data were analysed using FlowJo.

Geng G., Xu C., Peng N., Li Y., Liu J., Wu J., Liang J., Zhu Y, & Shi L. (2021). PTBP1 is necessary for dendritic cells to regulate T‐cell homeostasis and antitumour immunity. Immunology, 163(1), 74-85.

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10

Multicolor Flow Cytometry Staining

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Isolated cells were stained at 4°C in FACS buffer (PBS supplemented with 2.5% bovine serum albumin) with and hematopoietic lineage markers including Ly6G (BioLegend, clone 1A8, 1:600), CD11b (BioLegend, clone M1/70, 1:600) and Ter119 (BioLegend, clone TER-119, 1:600). This was followed by a second staining for NK1.1 (BioLegend, clone PK 136, 1:600), Thy1(CD90.2, BioLegend, clone 53–2.1 1:600), and Ly6C (BioLegend, clone HK1.4 1:600). Cell suspensions were labeled with DAPI just prior to flow cytometric analysis to allow exclusion of dead cells. Doublets, erythrocytes, and dead cells were excluded by forward scatter, Dapi, and Ter119. Neutrophils were identified as (Ter119^low/CD11b^high/Ly6G^high). Monocytes were identified as (Ly6G^low/Ter119^low/CD11b^high/Ly6C^high). NK or T cells were identified as (CD11b^low/Ly6G^low/(Nk1.1^high or CD90.2^high)) respectively. The Cre-induced fraction of each hematopoietic lineage subset was determined based on the fraction that was tdTomato^high. Data was acquired by Sony sorter MA900 at UCSD and analyzed with FlowJo software.

Taghdiri N., Calcagno D.M., Fu Z., Huang K., Kohler R.H., Weissleder R., Coleman T.P, & King K.R. (2021). Macrophage calcium reporter mice reveal immune cell communication in vitro and in vivo. Cell Reports Methods, 1(8), 100132.

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Clone 1a8

Lab products found in correlation

12 protocols using clone 1a8

Flow Cytometry and Immunohistochemistry Antibody Sources

Tumor-Infiltrating Lymphocyte Analysis

Adoptive Transfer of Tumor-Associated Monocytes

Macrophage Depletion in Mice for Immunological Studies

Adoptive Transfer of Tumor-Associated Monocytes

Multicolor Flow Cytometry Profiling of Hematopoietic Cells

BAL Cell Subset Identification

Intravital Microscopy of Leukocyte-Endothelial Interactions

Flow Cytometric Analysis of Immune Cells

Multicolor Flow Cytometry Staining

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