Pe rat anti mouse siglec f

Manufactured by BD

Sourced in United States

The PE rat anti-mouse Siglec-F is a fluorescently-labeled antibody that specifically binds to the mouse Siglec-F protein. Siglec-F is a sialic acid-binding immunoglobulin-like lectin that is expressed on the surface of eosinophils and plays a role in regulating eosinophil function.

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5 protocols using pe rat anti mouse siglec f

Multicolor Flow Cytometry of Lung Cells

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BAL was obtained using 3 × 0.7 ml cold PBS supplemented with 2% FCS (Biowest, Kansas City, MO) and 1% EDTA. Lungs were digested with Liberase CI (Roche). Both BAL and lung ceHs were stained with allophycocyanin anti-mouse Ly-6G (BioLegend, San Diego, CA), PE rat anti-mouse Siglec-F (BD Pharmingen), PetCP/Cy5.5 anti-mouse CDllc (BioLegend), and anti-mouse CD3 eEluor 450 (eBioscience). This staining panel distinguished neutrophils (Ly-6G⁺CD11c) from eosinophils (Siglec-F⁺CD11c⁻) and macrophages (Ly-6G⁻CD11c⁺).

Chua Y.L., Liong K.H., Huang C.H., Wong H.S., Zhou Q., Ler S.S., Tang Y., Low C.P., Koh H.Y., Kuo I.C., Zhang Y., Wong W.S., Peh H.Y., Lim H.Y., Ge M.Q., Haczku A., Angeli V., MacAry P.A., Chua K.Y, & Kemeny D.M. (2016). Blomia tropicalis–Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13–Dependent Mechanism. Journal of immunology (Baltimore, Md. : 1950), 197(10), 3771-3781.

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Granulocyte Identification in BALF

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For classification of granulocytes, cells from BALF were stained with Zombie Aqua™ solution for 10 min in the dark, stained for 30 min with antibodies for markers as follows: FITC Rat Anti-Mouse CD45 (#553079, BD, USA), PE/Cyanine7 anti-mouse CD11c (#117317, BioLegend, USA), BB700 Rat Anti-Mouse CD11b (#566416, BD, USA), APC Rat Anti-Mouse Ly-6G (#560599, BD, USA), PE Rat Anti-Mouse Siglec-F (#552126, BD, USA), BV421 Rat Anti-Mouse F4/80 (#565411, BD, USA). Absolute cell counts were calculated on the basis of Precision Count Beads^TM (#424902, BioLegend, USA). Data were collected on a BD Biosciences FACSVerse Flow Cytometer and analyzed using FlowJo software. The Flow cytometry gating strategy is shown in Supplementary Fig. S1F.

Ma L., He Y., Xie H., Wang J., Chen J., Song S., Zhang L., Li L., Lai H., Liu Y., Chen H., Zhang X., Liu X., Zou Z., Zhang Q., Yan J, & Tao A. (2024). Ferroptotic alveolar epithelial type II cells drive TH2 and TH17 mixed asthma triggered by birch pollen allergen Bet v 1. Cell Death Discovery, 10, 96.

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Isolation and Characterization of Murine Immune Cells

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Chicken egg ovalbumin (OVA, premium quality Grade V) was obtained from Sigma-Aldrich (St. Louis, MO). A TRPV4 KO mouse line on a C57BL/6 background was generated by Dr. Makoto Suzuki (Jichi Medical University, Tochigi, Japan) as described previously [22 (link)]. Inbred C57BL/6 mice (six-eight-week old) were obtained from Charles River (Wilmington, MA). Both male and female WT and TRPV4 KO mice were used in the experiments. The Institutional Animal Care and Use Committee (IACUC) of the University of Maryland reviewed and approved all animal research pertaining to this project, and all the animals were housed in HEPA filtered animal cages and provided ad libitum access to food and water.
APC-Cy7 rat anti-mouse CD45, APC-Cy7 rat IgG2b, FITC hamster anti-mouse CD11c, FITC Ar hamster IgG1, PE rat anti-mouse siglec-F, PE-rat IgG2a, APC-Cy7 rat anti-mouse Ly6G and Ly6C (Gr1), and APC-Cy7 rat IgG2b were purchased from BD Pharmingen (San Diego, CA); APC anti-mouse/human CD11b, APC rat IgG2b, and PE rat anti-mouse F4/80 were purchased from Biolegend (San Diego, CA).

Palaniyandi S., Rajendrakumar A.M., Periasamy S., Goswami R., Tuo W., Zhu X, & Rahaman S.O. (2019). TRPV4 is dispensable for the development of airway allergic asthma. Laboratory investigation; a journal of technical methods and pathology, 100(2), 265-273.

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Flow Cytometric Analysis of Immune Cells

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Cell suspensions were obtained from BALF and were washed and resuspended in PBS-1 %BSA before FASC analysis. All flow cytometry data was acquired using FACS Canto Ⅱ (BD) and data was analyzed using FlowJo 10.4. The following antibodies were used in flow cytometric analyses: BV421 Hamster Anti-Mouse TCR β Chain (BD Horizon, catalog no. 562839), APC-Cy™7 Rat Anti-Mouse CD4 (BD Pharmingen, catalog no. 565650), APC Rat Anti-Mouse CD8α (BD Pharmingen, catalog no. 553035), PE Mouse Anti-Mouse NK-1.1 (BD Pharmingen, catalog no. 557391), APC-Cy™7 Rat Anti-CD11b (BD Pharmingen, catalog no. 557657), FITC anti-mouse CD11c Antibody (Biolegend, catalog no. 117306), F4/80 Monoclonal Antibody APC (eBioscience, catalog no. 17-4801-80), PE Rat anti-Mouse CD14 (BD Pharmingen, catalog no. 553740), PE Rat Anti-Mouse Siglec-F (BD Pharmingen, catalog no. 552126).

Li Q., Li R., Yin H., Wang S., Liu B., Li J., Zhou M., Yan Q, & Lu L. (2022). Oral IRAK4 inhibitor BAY-1834845 prevents acute respiratory distress syndrome. Biomedicine & Pharmacotherapy, 153, 113459.

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Detecting Immune Cells in Tissues

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For the detection of monocytes, macrophages and neutrophils, the following fluorochrome-conjugated antibodies were used: anti-mouse CD11b FITC (eBioscience, 11-0112, AB_464933), anti-mouse Ly-6G (Gr-1) PE (eBioscience, 12-5931, AB_466045), anti-mouse F4/80 Antigen APC (eBioscience, 17-4801, AB_469452), and PE rat anti-mouse Siglec-F (BD Pharmingen™ 562068, AB_10896143). According to the reported methods, CD11b staining was used to detect monocytes, the combination of CD11b and Ly-6G (Gr-1) was used to detect neutrophils, and the combination of CD11b and Siglec-F was used to quantify eosinophils, while macrophages were detected with CD11b and F4/80. Both IECs and LPCs were combined for quantification of the immune cells. Total cells were resuspended in PBS containing 5% FBS, and at least 10⁶ cells were used for the staining.

Wei M., Ma Y., Shen L., Xu Y., Liu L., Bu X., Guo Z., Qin H., Li Z., Wang Z., Wu K., Yao L., Li J, & Zhang J. (2020). NDRG2 regulates adherens junction integrity to restrict colitis and tumourigenesis. EBioMedicine, 61, 103068.

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