Cd49b dx5

Manufactured by Thermo Fisher Scientific

CD49b (DX5) is a cell surface marker that is expressed on natural killer (NK) cells. It is commonly used in flow cytometry analysis to identify and study NK cell populations.

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Anti-CD49b (DX5; (6 citations)

12 protocols using cd49b dx5

Magnetic Isolation of Murine Lymphoid and Myeloid Cells

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Total splenocytes were incubated with biotin-conjugated antibodies against B220 (RA3-6B2), CD4 (GK1.5), CD8a (53-6.7) and CD49b (DX5) for lymphoid cells depletion, or with antibodies against CD11b (M1/70), CD11c (N418), I-A/I-E (M5/114.15.2), CD4 (GK1.5) and CD8a (53-6.7) (eBioscience, San Diego, CA) for myeloid and T cell elimination. Cells were washed and incubated with streptavidin-conjugated magnetic microbeads and negative selection was carried out in a MACS CS column as indicated by manufacturer (Miltenyi Biotec, Auburn, CA). T cells were purified from peripheral lymph node (LN) incubated with biotin-conjugated antibodies against B220 (RA3-6B2), Ly76 (TER-119), CD11b (M1/70), CD11c (N418), I-A/I-E (M5/114.15.2) and CD49b (DX5) (eBioscience). Cells were washed and incubated with Dynabeads Biotin Binder (Invitrogen, Oslo, Norway) following manufacturer instructions. T cells were >95% pure.

Galvani R.G., Lemos R., Areal R.B., Salvador P.A., Zamboni D.S., Wanderley J.L, & Bonomo A. (2015). Disease Severity and Mortality Can Be Independently Regulated in a Mouse Model of Experimental Graft versus Host Disease. PLoS ONE, 10(2), e0118079.

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Multiparametric Flow Cytometry Analysis

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Ears were collected and processed as described^{54 (link)}. All cells were first pre-incubated with anti-mouse CD16/CD32 for blockade of Fc γ receptors, then were washed and incubated for 40 min with the appropriate monoclonal antibody conjugates in a total volume of 200 μl PBS containing 2 mM EDTA and 2% (vol/vol) bovine serum. DAPI (Invitrogen) was used to distinguish live cells from dead cells during cell analysis. Stained cells were analyzed on a FACS Canto or LSRII machine using the Diva software (BD Bioscience). Data were analyzed with FlowJo software (TreeStar). The following fluorochrome-conjugated anti-mouse antibodies were used at indicated dilutions: CD103 (2E7, 1:200), CD11c (N418, 1:200), CD24 (30-F11, 1:200), CD11b (M1/70, 1:200), MHC-II (M5/114.15.2, 1:400), CD45 (30-F11, 1:200), CD64 (X54-5/7.1, 1:200), Ly6C (HK1.4, 1:200), TCRb (H57-597, 1:200), CD3 (145-2C11, 1:200), TCRgd (EBIOGL3, 1:200), B220 (RA3-6B2, 1:200), CD49b (DX5, 1:200) and FCeR1 (MAR-1, 1:200) were from eBioscience; Siglec-F (E50-2440, 1:200), Ly6G (1A8, 1:200), CD117 (2B8, 1:200), CD8 (53-6.7, 1:200), and CD4 (GK1.5, 1:200) were from BD Bioscience.

Chen L., Deshpande M., Grisotto M., Smaldini P., Garcia R., He Z., Gulko P.S., Lira S.A, & Furtado G.C. (2020). Skin expression of IL-23 drives the development of psoriasis and psoriatic arthritis in mice. Scientific Reports, 10, 8259.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Human single cell suspensions were surface stained with fluorescence conjugated mAb to CD134 (L106, BD Bioscience), CD137 (4B4-1, BD Bioscience), CD69 (FN50, eBioscience), CD56 (N901, Beckman Coulter), CD3 (SK7, eBioscience) and CD5 (OKT1, in house). Murine cells were first FcγR-blocked (2.4G2, in-house) and then stained with CD49b (DX5, eBioscience), NKp46 (29A1.4, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4–5, eBioscience), CD8 (53–6.7, eBioscience) or CD25 (PC61.5, eBioscience). For intracellular FOXP3 (NRRF-30, eBioscience) staining, cells were fixed and permeabilised as per manufacturer’s protocol (eBioscience). Acquisition was performed using FACSCalibur (BD Biosciences) and analysed using Cytobank (Cytobank).

Turaj A.H., Cox K.L., Penfold C.A., French R.R., Mockridge C.I., Willoughby J.E., Tutt A.L., Griffiths J., Johnson P.W., Glennie M.J., Levy R., Cragg M.S, & Lim S.H. (2018). Augmentation of CD134 (OX40)-dependent NK anti-tumour activity is dependent on antibody cross-linking. Scientific Reports, 8, 2278.

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Isolation and Enrichment of Innate Lymphoid Cells

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Lungs were perfused with PBS, diced into ~2-cm pieces and incubated with Liberase TM (42.4 μg/ml) and DNAse I (10 U/ml; both from Roche) for 45 min at 37 °C before being mashed through a 70-μM cell strainer and washed with complete RPMI. Remaining blood cells were lysed with ACK cell lysing buffer (Invitrogen) and single cell suspensions were incubated with biotinylated antibodies to CD3ε (clone 145-2C11), CD19 (1D3), B220 (RA3-6B2), CD5 (53-7.3), TCRβ (H57-597), TCRγδ (GL3), CD11c (N418), F4/80 (BM8), Gr-1 (RB6-8C5), Ter119 (TER-119), CD49b (DX5; all eBioscience) and CD27 (LG3A10, BioLegend) all at 25 μg/ml per 2 × 10⁸ cells. Cells were then incubated with anti-biotin microbeads (Miltenyi; 1:5 dilution at 10⁸ cells per ml) and depleted following manufacturer’s protocol. For each ILC enrichment, the depletion was repeated twice and yielded >95% pure ILC populations (Supplementary Fig. 3a).

Silver J.S., Kearley J., Copenhaver A.M., Sanden C., Mori M., Yu L., Pritchard G.H., Berlin A.A., Hunter C.A., Bowler R., Erjefalt J.S., Kolbeck R, & Humbles A.A. (2016). Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nature immunology, 17(6), 626-635.

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Multiparameter Flow Cytometry Immunophenotyping

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The following antibodies were used: CD11b-APC-Cy7 (M1/70), CD19-Biotin (6D5), TCRβ-Biotin (H57-597), NK1.1-Biotin (PK136), Ly6C-BV570 (HK1.4), Ly6G-PerCp-Cy5.5 (1A8), and TCRβ-Pacific Blue (H57-597) from Biolegend (San Diego, CA) CD8α-PE-Cy7 (53-6.7), CD4-Alexa 700 (RM4-5), and CD19-FITC (1D3) from BD Pharmingen (San Diego, CA); CD49b (DX5) from eBioscience (San Diego, CA) and MHC-II-Biotin (produced in house from hybridoma M5/114). The secondary Streptavidin-PE-CF594 from BD Horizon was used with biotin-conjugated primary antibodies. Antibody for detection of intracellular IFN-γ was IFN-γ-APC (XMG1.2) and the isotype control was Rat IgG1, κ-APC (RTK2071) (both from Biolegend). PE-labeled CD1d-tetramers loaded with α-galactosyl ceramide (αGalCer) (PBS57) were provided by the NIH Tetramer Facility and used to detect NKT cells.

Fernández-Santoscoy M., Wenzel U.A., Yrlid U., Cardell S., Bäckhed F, & Wick M.J. (2015). The Gut Microbiota Reduces Colonization of the Mesenteric Lymph Nodes and IL-12-Independent IFN-γ Production During Salmonella Infection. Frontiers in Cellular and Infection Microbiology, 5, 93.

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Multiparametric Flow Cytometry of Immune Cells

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Antibodies against the following antigens were used: CD11b (M1/70; eBioscience), CD49b (DX5; eBioscience), Siglec-F (E50-2440; BD), Ly6G/C (RB6-8C5; BD), FcεRI (Mar-1; BioLegend), CD45 (30-F11; BioLegend), CD34 (RAM34; eBioscience), ESAM-1 (1G8; BioLegend), CD31 (390; eBioscience), c-kit (2B7; BioLegend), CD106 (429; eBioscience), IL-4Rα (mIL4-M1; BD), and CD213a1 (13MOKA; eBioscience). Key cell populations were defined as follows: (a) eosinophils, FSC^loSSC^hiCD11b⁺Siglec-F⁺ or FSC^loSSC^hiSiglec-F⁺4get⁺; (b) basophils, FSC^loSSC^loCD49b⁺FcεRI⁺Gr-1⁻CD4⁻CD8⁻CD19⁻γδ⁻Siglec-F⁻ or FSC^loSSC^loBasoph8⁺CD49b⁺; (c) mast cells, 4get⁺ckit⁺ or FSC^hiSSC^hic-kit⁺CD11a⁻CD44⁺; (d) endothelial cells, CD45⁻CD34⁺ESAM-1⁺; and (e) PMNs/monocytes, Gr-1⁺CD11b⁺. Flow cytometry data acquisition was performed on an LSRII (BD), using FlowJo to analyze the data.

Cheng L.E., Sullivan B.M., Retana L.E., Allen C.D., Liang H.E, & Locksley R.M. (2015). IgE-activated basophils regulate eosinophil tissue entry by modulating endothelial function. The Journal of Experimental Medicine, 212(4), 513-524.

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Isolation of Mouse Neutrophils

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Negative selection of neutrophils from whole bone marrow cells was performed by Magnetic Cell Seperation (MACS; Miltenyi Biotec) according to a previously published protocol, which allows isolation of highly purified primary untouched mouse neutrophils [21 (link)]. Briefly, bone marrow cells were flushed from the femur of mice and stained with the following anti-mouse antibodies (all biotinylated): CD5 (BD Biosciences), CD45R/B220 (Biolegend), CD49b/DX5 (eBiosciences), CD117 (eBiosciences), F4/80 (eBiosciences) and Ter 119 (Biolegend). After incubation the unbound antibodies were washed away. Bone marrow cells were then incubated with magnetic beads (MACS, Miltenyi Biotec, Germany) labeled with streptavidin. Bead coupled bone marrow cells were removed by immunomagnetic separation following the manufacturer’s recommendation resulting in highly purified neutrophils.

Carevic M., Öz H., Fuchs K., Laval J., Schroth C., Frey N., Hector A., Bilich T., Haug M., Schmidt A., Autenrieth S.E., Bucher K., Beer-Hammer S., Gaggar A., Kneilling M., Benarafa C., Gao J.L., Murphy P.M., Schwarz S., Moepps B, & Hartl D. (2016). CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense. Journal of Innate Immunity, 8(4), 362-373.

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Purification of γδ T Cell Subsets

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After blocking with anti-CD16/32, cells were negatively selected from pooled pLN and splenocytes using anti-TCRβ biotin, anti-CD45R biotin (RA3-6B2), and anti-CD11b (M1/70) antibodies together with MagniSort™ Streptavidin Negative Selection Beads according to the manufacturer’s instructions (eBioscience). The negatively selected cells were then stained with fluorochrome-labeled anti-γδTCR, anti-CD27, and/or anti-Vγ4 antibodies. γδ subsets (Vγ4^{− γδ27+}, Vγ4+ γδ27+, Vγ4^{− γδ27−}, and Vγ4^{+ γδ27−}) were sorted using an Aria II with a purity of at least 98%. Alternatively, total γδ cells were manually isolated by negative selection as described previously (30 (link)) with modification. Biotin-conjugated mAbs against the following proteins were used: TCRβ, CD4, CD8α, CD45R, CD11b (M1/70), CD11c, Ly-6G (RB6-8C5), TER-119 (TER-119), and CD49b (DX5) (all from eBioscience).

Kim G., Gu M.J., Kim S.J., Ko K.H., Kye Y.C., Kim C.G., Cho J.H., Lee W.K., Song K.D., Chu H., Park Y.M., Han S.H, & Yun C.H. (2018). Transcription Factor KLF10 Constrains IL-17-Committed Vγ4+ γδ T Cells. Frontiers in Immunology, 9, 196.

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Lung Immune Cell Isolation and Analysis

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Lung tissues were diced and subsequently digested for 45 min at 37 °C with 5 mg/ml Liberase^TM (Sigma, St Louis, MO, USA) and 10 mg/ml DNase (Roche, Basel, Switzerland). Single‐cell suspensions were stained and analyzed using a FACS Canto II cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Antibodies were purchased from BioLegend unless otherwise listed. For monocytes and neutrophils, the following antibodies were used: Ly6G (1A8), CD45 (30‐F11; eBioscience, San Diego, CA, USA), CD11c (HL3; BD, San Jose, CA, USA), CD11b (M1/70; BD), Ly6C (AL‐21; BD), and IL1RL1 (DIH9). For ILC staining, the following antibodies were used: CD3 (145‐2C11), CD19 (6D5), Gr1 (RB6‐8C5), B220 (RA3‐6B2), Ter119 (TER‐119), FcaR1 (MAR‐1), CD49b (DX5; eBioscience), Il1rl1 (DIH9), CD90 (30‐H12), and CD45 (30‐F11). A Fixable Viability Dye kit (eBioscience) was used to exclude dead cells. Data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).

Ramirez‐Moral I., Blok D.C., Bernink J.H., Garcia‐Laorden M.I., Florquin S., Boon L., van't Veer C., Mack M., Saluzzo S., Knapp S., Spits H., de Vos A.F, & van der Poll T. (2021). Interleukin‐33 improves local immunity during Gram‐negative pneumonia by a combined effect on neutrophils and inflammatory monocytes. The Journal of Pathology, 253(4), 374-383.

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Immunophenotyping Bone Marrow Cell Subsets

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BM cell suspensions were prepared, stained, and analyzed as described previously (Arndt et al., 2013 (link)). Antibodies (clones in parentheses) used are as follows: CD3 (2C11; 17A2), CD11b (M1/70), CD19 (1D3), CD34 (RAM34), CD45.1 (A20), CD45.2 (104), CD45R (RA3-6B2), CD86 (GL1), CD117 (2B8), CD135 (A2F10), Gr-1 (RB6-8C5), Nk1.1 (PK136), Sca1 (D7), Ter119 (Ter119), CD41 (MWReg30), Epcr (1550), CD49b (DX5), and Ifnr1 (MAR1-5A3; all eBioscience); CD48 (HM48-1) and CD150 (TC15-12F1; BioLegend); and Tgfbr2 (polyclonal; R&D Systems). MFI of Kit receptor expression was normalized between independent experiments based on the MFI of Kit expression on wild-type LS CD48⁻ CD150⁺ cells: (MFI Kit on donor-derived cells in experiment 1) = (MFI Kit on wild-type cells in experiment 1)/(MFI Kit on wild-type cells in experiment 2) × (MFI Kit on donor-derived cells in experiment 2).

Grinenko T., Arndt K., Portz M., Mende N., Günther M., Cosgun K.N., Alexopoulou D., Lakshmanaperumal N., Henry I., Dahl A, & Waskow C. (2014). Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells. The Journal of Experimental Medicine, 211(2), 209-215.

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