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Anti mouse lineage cocktail

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Anti-mouse lineage cocktail is a reagent used to identify and exclude lineage-positive cells, such as T cells, B cells, monocytes, granulocytes, and natural killer cells, in flow cytometric analysis. It is designed to facilitate the identification and analysis of non-lineage-positive cells, such as stem cells or progenitor cells, in mouse samples.

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Lab products found in correlation

Facscanto 2, by BD (2 mentions) Facsdiva software, by BD (2 mentions) Anti cd16 32, by BioLegend (2 mentions) Anti cd45, by BioLegend (2 mentions) Transcription factor staining kit, by Thermo Fisher Scientific (1 mentions) Anti cd16 32, by Thermo Fisher Scientific (1 mentions) Nhs fluorescein, by Thermo Fisher Scientific (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Anti mouse cd11c, by BioLegend (1 mentions) Anti mouse f4 80, by BioLegend (1 mentions) Anti cd16 cd32, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Anti nk1, by BioLegend (1 mentions) Anti cd8α, by BioLegend (1 mentions) Anti tnf mp6 xt22, by BioLegend (1 mentions) Anti ifn γ, by BD (1 mentions) Anti f4 80, by BioLegend (1 mentions) Anti b220, by BioLegend (1 mentions) Anti c kit, by BioLegend (1 mentions) Anti sca 1, by BioLegend (1 mentions)

Close spelling variants of this product

FITC anti-mouse Lineage Cocktail BV421™ anti-mouse lineage cocktail Lineage Cocktail

6 protocols using anti mouse lineage cocktail

1

Characterization of Lung Immune Responses in WT and AhR-/- Mice

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The lungs from P. brasilienis infected WT and AhR−/− mice were collected after 96 h, 2 and 10 weeks of infection, digested enzymatically and lung leukocytes prepared as previously described39 (link). For cell-surface staining, lung cells were suspended at 1 × 106 cells/mL in staining buffer. Fc receptors were blocked with unlabeled anti-CD16/32 (eBioscience) and then stained for 30 min on ice with fluorophore-conjugated antibodies. For myeloid cells the following antibodies were used: anti-CD11c, CD40, CD80, CD86 and MHC-II (IAb+). For lymphocytes: anti-CD4, CD25, CD8, CD44, and CD62L. For ILCs characterization, lung leukocytes were first treated with anti-mouse lineage cocktail (Biolegend) containing antibodies to CD3, Ly6G/Ly6C, CD11b, CD45R/B220, TER 119/erytroid cells, that react with T cells, B cells, monocytes, macrophages, NK cells and erythrocytes. Intracellular staining was conducted using the eBioscience Transcription Factor staining kit and specific antibodies for IL-17, IL-4, IFN-γ, IL, 22, IL-1β, IL-12, TNF-α, IL-6, TGF-β, IL-10, FoxP3, IDO-1 and AhR. Cells were run on FACSCantoII (BD Biosciences) and a minimum of 50,000 events was acquired using a FACSDiva software (BD Biosciences). Cells were analyzed using FlowJo software (Tree Star).
de Araújo E.F., Preite N.W., Veldhoen M., Loures F.V, & Calich V.L. (2020). Pulmonary paracoccidioidomycosis in AhR deficient hosts is severe and associated with defective Treg and Th22 responses. Scientific Reports, 10, 11312.
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2

Immune Cell Phenotyping Protocol

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Live/dead cells were discriminated using with eFluor 780 fixable viability dye (eBioscience,) for 30 mins at room temperature covered from light. Cells were then blocked with anti-CD16/CD32 (eBioscience) for 20 min. Cells were subsequently stained for extracellular markers anti-mouse CD11b (30-H12), anti-mouse CD45 (30-F11), anti-mouse Ly6G (1A8), anti-mouse CD3 (145-2C11), anti-mouse CD4 (L3T4), anti-mouse CD11c (N418) anti-mouse F4/80 (BM8), anti-mouse lineage cocktail (Biolegend, 133302), anti-mouse Sca-1 (D7), anti-mouse ST1 (D1H9), anti-mouse CD127 (A7R34), anti-mouse 90.2 (30-H12), anti-mouse CD25 (3C7), anti-mouse Siglec-F (S17007L), anti-mouse CD8α (25–0081) for 30 minutes. Samples were fixed for 15 min with 2% PFA. Sample acquisition was conducted using BD LSRFortessa and analysed using FlowJo Software. The IL-4R neutralizing antibody (kindly provided from of Dr. Manel Jordana, McMaster University) was labelled with NHS-Fluorescein (Thermo Scientific).
Lee A.J., Feng E., Chew M.V., Balint E., Poznanski S.M., Giles E., Zhang A., Marzok A., Revill S.D., Vahedi F., Dubey A., Ayaub E., Jimenez-Saiz R., McGrath J.J., Ritchie T.M., Jordana M., Jonigk D.D., Ackermann M., Ask K., Miller M., Richards C.D, & Ashkar A.A. (2022). Type I interferon regulates proteolysis by macrophages to prevent immunopathology following viral infection. PLoS Pathogens, 18(5), e1010471.
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3

Detailed Antibody Characterization for Immunoblotting and Flow Cytometry

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Primary antibodies used for immunoblotting were: anti-Snrnp40 (HPA026527) from Atlas; anti-FLAG M2 (F1804) from Sigma-Aldrich; anti-Themis (06–1328) and anti-GAPDH (MAB374) from Millipore; anti-Snrnp200 (A303–453A-T) from Bethyl; anti-Eftud2 (10208–1-AP) from Proteintech; anti-Cd2bp2 (PA5–18286) from Invitrogen; anti-Ikzf3 (Aiolos, 15103), anti-IL-17A (13838), anti-IL-17F (13186), anti-α-tubulin (3873), anti-β-actin (3700), anti-Hsp90 (4874) and anti-GFP (2956) from Cell Signaling. Antibodies used for flow cytometry were: anti-CD3ε (145–2C11), anti-CD4 (RM4–5), anti-CD8α (53–6.7), anti-B220 (RA3–6B2), anti-NK-1.1 (PK136), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD11c (HL3), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-mouse Lineage Cocktail (CD3/Ly-6G/CD11b/B220/Ter-119), anti-c-Kit (CD117, 2B8), anti-Sca-1 (Ly-6A/E, D7), anti-IL-7Rα (CD127, SB/199), anti-CD16/32 (93), anti-Flk-2 (CD135, A2F10), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD107a (Lamp-1, 1D4B), anti-TNF (MP6-XT22) from BioLegend or BD Biosciences, and anti-CD34 (RAM34), anti-granzyme B (NGZB), anti-IFN-γ (XMG1.2) from eBioscience.
Zhang D., Yue T., Choi J.H., Nair-Gill E., Zhong X., Wang K.W., Zhan X., Li X., Choi M., Tang M., Quan J., Hildebrand S., Moresco E.M, & Beutler B. (2019). Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40. Nature immunology, 20(10), 1322-1334.
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4

Murine Hematopoietic Stem Cell Isolation

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Female C57BL/6 (B6) and BALB/c mice at 8–10 weeks of age were purchased from SLAC Ltd. Animals were maintained in the Guangxi Normal University Laboratory Animal Center and handled in accordance with the institution’s guidelines. All experimental protocols were approved by the Guangxi Animal Management Committee of Guangxi S&T Department (Project Number syxk (Gui) 2013-0001). Fluorescence-conjugated anti-mouse mAbs anti-c-kit(ACK2), anti-Sca-1(D7), anti-CD34(RAM34), anti-IL-7R(A7R34), anti-FcR II/III(93), anti-Flt3(A2F10), anti-Thy-1.1(HIS51), and their isotype ctrl antibodies (except those for anti-IL-7R and anti-Thy-1.1) were obtained from affymetrix eBioscience. Anti-mouse Lineage cocktail (containing anti-CD3, Ly-6G/Ly-6C, anti-CD11b, anti-CD45R/B220 and anti-TER-119) with isotype ctrl was from Biolegend. Mitomycin C was obtained from Solarbio.
Pu S., Qin B., He H., Zhan J., Wu Q., Zhang X., Yang L., Qu C, & Zhou Z. (2016). Identification of early myeloid progenitors as immunosuppressive cells. Scientific Reports, 6, 23115.
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5

Multiparametric Flow Cytometry Analysis of Lung Immune Cells

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Lung cell suspensions were adjusted to 1x106 cells and suspended in PBS-azide (0.1%) containing fetal bovine serum (SFB, 5%). Fc receptors were blocked with anti-CD16/32 monoclonal antibody and then labeled with fluorophore-conjugated antibodies as previously described (37 (link)) Labeled antibodies (BD Biosciences) were used in the appropriate combination for the cell population to be analyzed. For lymphocytes, the following antibodies were used: anti-CD3, CD4, CD25, and Foxp3; for myeloid cells: anti-CD45, CD11b, CD11c, CD40, CD80, CD86, MHC-II, and F4/80. For ILCs characterization, lung leukocytes were first treated with an anti-mouse lineage cocktail (Biolegend) containing antibodies to CD3, Ly6G/Ly6C, CD11b, CD45R/B220, TER 119/erythroid cells, that react with T cells, B cells, monocytes, macrophages, NK cells, and erythrocytes. Intracellular staining was conducted using the eBioscience Transcription Factor staining kit and specific antibodies for IL-17, IL-4, IFN-γ, IL-22, IL-1β, IL-12, TNF-α, IL-6, TGF-β, IL-10, FoxP3, IDO-1, and AhR. Supplementary Table 1 lists the monoclonal antibodies used in flow cytometry assays. Cells were run on FACSCantoII (BD Biosciences) and a minimum of 50,000 events was acquired using FACSDiva software (BD Biosciences). Cells were analyzed using FlowJo software (Tree Star).
de Araújo E.F., Loures F.V., Preite N.W., Feriotti C., Galdino N.A., Costa T.A, & Calich V.L. (2021). AhR Ligands Modulate the Differentiation of Innate Lymphoid Cells and T Helper Cell Subsets That Control the Severity of a Pulmonary Fungal Infection. Frontiers in Immunology, 12, 630938.
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6

Murine Hematopoietic Stem Cell Expansion

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WT mouse BM was harvested and lineage depleted (Milltenyi
130–110-470). Lineage depleted cells were grown for 4 days in liquid
culture (RPMI1640 supplemented with 10%FBS and 100ng/mL recombinant murine
(rmu)SCF, rmuTPO, rhuFLT3L (FL)) with or without 20ng/mL recombinant CXCL15.
Cells were collected on Day 0 and Day 4 and plated for colony formation analysis
as described in the previous section. Cells were collected on Day 0 and Day 4
and stained with fluorophore conjugated anti-mouse lineage cocktail, anti-CD117,
anti-Ly6A/E, anti-CD34, anti-CD135, anti-CD150, and anti-CD16/32 (BioLegend).
Numbers of HSCs/HPCs, etc. were determined by flow cytometry.
Broxmeyer H.E., Cooper S.H, & Ropa J. (2021). CXCL15/Lungkine has Suppressive Activity on Proliferation and Expansion of Multi-potential, Erythroid, Granulocyte and Macrophage Progenitors in S-Phase Specific Manner. Blood cells, molecules & diseases, 91, 102594.
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