Anti mouse cd4

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-mouse CD4 is a laboratory reagent used to identify and isolate CD4-positive cells in mouse samples. It functions as a specific antibody that binds to the CD4 surface marker on T helper cells. This product can be used in various cell analysis and sorting applications.

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Anti-CD4 (178 citations) Anti-mouse CD4 fluorescein isothiocyanate (FITC, (7 citations) PE-Cy5 conjugated anti-mouse CD4 (6 citations) Anti-mouse CD4-eFluor450 (3 citations) Monoclonal anti-mouse CD3 (Pe-Cy5-labeled) and CD4 (FITC-labeled) antibodies (2 citations) Anti-mouse CD4-PerCP-Cy5.5 (2 citations) FITC)-conjugated anti-mouse CD4 (clone GK1.5; (2 citations) FITC)-conjugated anti-mouse CD4 antibody (2 citations) PE-Cy7-conjugated anti-mouse CD4 (RM4–5, (2 citations) FITC-conjugated anti-mouse CD4 monoclonal antibody (mAb), (2 citations)

27 protocols using anti mouse cd4

Peripheral Blood Lymphocyte Immunophenotyping

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Anti-Mouse CD8a and Anti-Mouse CD4, labelled with phycoerythrin (PE) and PerCP-Cy5.5, respectively, were purchased from eBioscience (San Diego, CA, USA) Each test contained 50 µL of peripheral blood sample, and then incubated 0.25 µL PE labeled Anti-Mouse CD8a and 0.25 µL PerCP-Cy5.5 labeled Anti-Mouse CD4. After incubating for 20 min in the dark, 2 mL lysing solution (Becton Dickinson, Franklin Lakes, NJ, USA, 10%, v/v) was added to make the lysis of erythrocytes more complete and surviving cells were washed twice with PBS, with centrifugation between each step for 5 min at 500 g. FACScan flow cytometer was used for FCM analysis (BD Biosciences, San Jose, CA, USA). First, an electronic gate was set on lymphocytes and then gated CD4+ T cells in Y axis and CD8+ cells as figure. Dead cells were excluded according to the forward scatter and side scatter.

Peng J., Xiao Y., Wan X., Chen Q., Wang H., Li J., Chen J, & Gao R. (2020). Enhancement of Immune Response and Anti-Infection of Mice by Porcine Antimicrobial Peptides and Interleukin-4/6 Fusion Gene Encapsulated in Chitosan Nanoparticles. Vaccines, 8(3), 552.

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Flow Cytometry Analysis of T Cell Subsets

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BD LSRFortessa X-20 flow cytometer (BD Biosciences) and FlowJo software (Treestar) were used for acquisition and analysis, respectively. The antibodies used include anti-mouse CD8 (#563786), anti-mouse CD25 (#558642), anti-mouse TCRβ (#553174) from BD Biosciences; anti-mouse CD4 (#100544) from BioLegend; anti-mouse CD3 (#14-0031-86), anti-mouse CD4 (#17-0042-83), anti-mouse CD4 (#17-0041-83) and anti-mouse CD69 (#25-0691-82) from eBioscience. Kinase inhibitor library was from Cayman Chemical (#10505).

Chen E.W., Brzostek J., Gascoigne N.R, & Rybakin V. (2018). Development of a screening strategy for new modulators of T cell receptor signaling and T cell activation. Scientific Reports, 8, 10046.

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Cytokine and Antibody Protocols for in vitro Experiments

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All recombinant cytokines in in vitro experiments were obtained from Peprotech (London, UK). The antibodies in this study were purchased from commercial companies. Anti-mouse CD4, anti-mouse IFN-γ, anti-mouse CD25, anti-mouse Foxp3, anti-mouse CD11c, anti-mouse CD80, anti-mouse CD86, anti-mouse MHC-II, anti-mouse CD103, anti-human CD4, anti-human IFN-γ, anti-human CD25, anti-human Foxp3, anti-human CD11c, anti-human CD80, anti-human CD86, anti-human CD83, and anti-human CD103 were obtained from ebioscience (CA, USA). Anti-mouse IDO and anti-human IDO were purchased from Biolegend (CA, USA) and R&D (MN, USA), respectively.

Tu L., Chen J., Zhang H, & Duan L. (2017). Interleukin-4 Inhibits Regulatory T Cell Differentiation through Regulating CD103+ Dendritic Cells. Frontiers in Immunology, 8, 214.

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Detailed Immunological Antibody Protocols

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The antibodies used in this study for flow cytometry include: anti-mouse CD8 (#563786), anti-mouse CD25 (#558642), anti-mouse TCRβ (#553174), anti-human CD3 (#563798), anti-human CD8 (#563795), and anti-human TCRαβ (#563826) from BD Biosciences; anti-mouse CD4 (#100544), anti-mouse IFN-γ (#505835), anti-human LFA-1 (#363404), anti-human CD11a (#301208), anti-human CD18 (#302114), anti-human CD25 (#302625), and anti-human CD69 (#310920) from BioLegend; anti-mouse CD4 (#17-0042-83), anti-mouse CD4 (#17-0041-83), anti-mouse CD69 (#25-0691-82), anti-mouse TNF (#17-7321-82), anti-mouse IL-2 (#12-7021-82), anti-human CD4 (#17-0048-2), and anti-human CD8 (#11-0088-41) from eBioscience.
The antibodies used for immunoprecipitation and immunoblotting include: anti-Cdc7 (#ab10535), anti-Dbf4 (#ab124707), anti-RNA polymerase II (#ab817) and anti-RNA polymerase II (phospho S2, # ab193468) from Abcam; anti-PLCγ (#610027) and anti-ERK (#610031) from BD Biosciences; anti-LAT (#641102) from BioLegend; anti-GAPDH (#2118), anti-pPLCγ (#2821), anti-ZAP70 (#2709), anti-pZAP70 (#2717), anti-pERK (#4370), anti-pLAT (#3584), anti-pLck (#6943), and anti-NF-κB (#3035) from Cell Signaling; anti pMCM2 S40 (#GTX62847) from GeneTex; anti-MCM2 (#MA5-15895) from Pierce; and anti-Lck (#sc-433) from Santa Cruz.

Chen E.W., Tay N.Q., Brzostek J., Gascoigne N.R, & Rybakin V. (2019). A Dual Inhibitor of Cdc7/Cdk9 Potently Suppresses T Cell Activation. Frontiers in Immunology, 10, 1718.

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Immune Cell Profiling by Flow Cytometry

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The spleen tissue and single cell suspensions were prepared as described above in cell staining buffer (BioLegend, Inc., San Diego, CA, USA). The debris was removed by filtration of the cell suspension through 70 µm nylon mesh strainer. Viable cells were counted and suspended in cell staining buffer at 1×10⁷ cells/ml. Cell suspension (100 µl) was distributed into aseptic Eppendorf plastic tubes. Cells were blocked with PBS containing 1% BSA. Isotype controls of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated anti-mouse IgG2b were used. FITC 0.25 µg anti-mouse CD4 and 0.25 µg PE anti-mouse CD8 (eBioscience, Inc.) per million cells in a 100 µl total staining volume were added, then incubated in the dark at 4°C for 20 min. Cells were washed twice and the cell pellets were resuspend in 0.5 ml cell staining buffer for analyzes by flow cytometry (FACSCalibur; BD Biosciences). A total of 10,000 events per test were collected.

Liu W., Xu Y., Shen H., Yan J., Yang E, & Wang H. (2017). Recombinant Bacille Calmette-Guérin coexpressing Ag85B-IFN-γ enhances the cell-mediated immunity in C57BL/6 mice. Experimental and Therapeutic Medicine, 13(5), 2339-2347.

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Alloantigen-Induced Lymph Node Expansion

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Male Balb/c mice (8-10 weeks-old) were given foot pad injections in the right hind limb of 1×10⁷ splenocytes from male DBA-2 mice (8-10 weeks-old). Recipient mice were immediately treated as follows (schematic in Figure 8A)
On day 4 after alloantigen challenge, the mice were sacrificed and the draining ipsilateral popliteal LNs (IL-pLNs) were collected. Following mechanical disruption of the pLNs, isolated cells were counted on a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA) to determine LN cellular expansion due to alloresponses. From each IL- pLN cell suspension, 1×10⁶ cells were stained for flow cytometry using LIVE/DEAD fixable near infra-RED and antibodies against the following surface markers: anti-mouse CD3, anti-mouse CD8, anti-mouse CD62L (BD Bioscience), anti-mouse CD4 (e-Bioscience), anti-mouse CD44 and anti-mouse CD45 (Biolegend). Samples were evaluated using a Cytoflex flow cytometer and data analyzed with Flow Jo.

Velluto D., Bojadzic D., De Toni T., Buchwald P, & Tomei A.A. (2020). Drug-Integrating Amphiphilic Nanomaterial Assemblies: 1. Spatiotemporal Control of Cyclosporine Delivery and Activity Using Nanomicelles and Nanofibrils. Journal of controlled release : official journal of the Controlled Release Society, 329, 955-970.

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Multiparameter Flow Cytometry Analysis

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Fluorescein isothiocyanate (FITC), Allophycocyanin cyanine tandem (APC-H7), R-phycoerythrin (PE) or Allophycocyanin (APC)-conjugated monoclonal antibodies (mAbs) were used for cytofluorometric analysis of anti-mouse Ki67 (BD PharMingen, San Diego, CA, USA), anti-mouse CD4, anti-mouse CD25 and anti-mouse FoxP3 (eBioscience, San Diego, CA). Purified hamster anti-mouse mAbs, anti-CD3 (clone 145-2C11) and anti-CD28 (clone 37.51) were also purchased from BD Pharmingen. Recombinant TGF-β was purchased from Peprotech (NJ, USA). TGF-β neutralizing mAb (1D11) was a gift from Dr. Hong-Ming Hu (Earle A. Chiles Research Institute, Portland, OR). Cell enrichment kits for CD4⁺ and Antigen Presenting Cells (APC, CD90.1⁻) were purchased from MACS Miltenyi Biotec Inc., (Auburn, CA, USA). Dead Fixable Violet Dead Cell Stain Kit was purchased from Invitrogen (L34955, Carlsbad, CA).

Polanczyk M.J., Walker E., Haley D., Guerrouahen B.S, & Akporiaye E.T. (2019). Blockade of TGF-β signaling to enhance the antitumor response is accompanied by dysregulation of the functional activity of CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ T cells. Journal of Translational Medicine, 17, 219.

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Multiparametric Flow Cytometry Panel

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Anti-mouse CD3 (eFlour450,17A2), anti-mouse CD4 (APC-eFlour780, RM4-5), anti-mouse CD8a (AF700, 53-6.7), anti-mouse CD11c (percp-cy5.5,N418), CD80 (PE-cy7, 16-10A1), CD86 (APC-cy7, GL1), anti-MHC class I (PE, SF1-1.1.1), anti-MHC class II (APC, AMS-32.1), anti-IFN-γ (APC, XMG1.2), anti-mouse TNF-α (PE-cy7, MP6-XT22), OVA_257-264 (SIINFEKL) peptide bound to H-2Kb monoclonal Ab (mAb) (PE-cy7, 25-D1.16), anti-mouse CD45.1 (PE, A20), and anti-mouse CD45.2 (AF700, 104) were from eBioscience. CD11c (APC, 3.9, #301613); CD80 (PE, 2D10, #207831); CD83 (BV421, HB15e, #147674); CD86 (PE-cy7, BU63, #202906); anti-HLA-A, B, C (BV605, W6/32, #212641); anti-HLA-DR, DP, DQ (Percpcy5.5, Tü39, #211013); anti-CD1c (APC/Fire750, L161, #331510); and anti-CD141 (BV785, M80, #344112) were obtained from BioLegend (San Diego, CA).

Wu J., Yang H., Xu J.C., Hu Z., Gu W.F., Chen Z.Y., Xia J.X., Lowrie D.B., Lu S.H, & Fan X.Y. (2021). Mycobacterium tuberculosis Rv3628 isan effective adjuvant via activationof dendritic cells for cancer immunotherapy. Molecular Therapy Oncolytics, 23, 288-302.

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Multiparameter Flow Cytometry Profiling

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All cells were stained with Fixable Viability dye (eBioscience, Biolegend) and treated with Fc block (eBioscience) prior to staining with fluorochrome-conjugated anti-mouse mAbs. mAbs were from Biolegend (F4/80, Ly6C, Ly6G, IgD, CD19, CD86, CD80, IFNAR, IgG1) and eBioscience (CD11b, MHCII, IgM).
For intracellular cytokine staining, cells were stimulated with PMA (Sigma-Aldrich), ionomycin (Sigma-Aldrich) and monensin (BD Biosciences) for 4 hours. Cells were then stained with anti-mouse CD4 (eBioscience), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained for IL-17 (BioLegend) and GM-CSF (Biolegend). All flow cytometric data were collected on LSRII (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.).
For intranuclear staining of Ki-67, cells were stained with anti-mouse CD4 (BDBioscience), fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer Set and stained for Ki-67 (Biolegend) to assess for cellular proliferation.

Agasing A.M., Wu Q., Khatri B., Borisow N., Ruprecht K., Brandt A.U., Gawde S., Kumar G., Quinn J.L., Ko R.M., Mao-Draayer Y., Lessard C.J., Paul F, & Axtell R.C. (2020). Transcriptomics and proteomics reveal a cooperation between interferon and T-helper 17 cells in neuromyelitis optica. Nature Communications, 11, 2856.

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Th1/Th2 Differentiation of CD4+ T Cells

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Purified CD4+T cells (CD4⁺CD25⁻CD45RB^hi) from spleens and lymph nodes of Mina KO or WT litter mate control mice were FACS sorted on a Reflection (i-Cyt, Champaign, IL) following staining with anti-mouse CD4, anti-mouse CD45RB and anti-mouse CD25 (eBioscience) were differentiated in to Th1 or Th2 as per previously published conditions [54 (link)]. Briefly, CD4+T cells were stimulated in anti-CD3 coated plates (5ug/ml), anti-CD28 (1ug/ml) in presence of rIL12 (20ng/ml) and anti-IL4(10ng/ml) for Th1 and anti-IFNγ (10ng/ml), rIL4 (100ng/ml) and anti-IL12 (10ng/ml) for Th2 differentiation. Cells were cultured for three days and restimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) in presence of monensin to test for intracellular cytokine IFNγ and IL4.

Pillai M.R., Mihi B., Ishiwata K., Nakamura K., Sakuragi N., Finkelstein D.B., McGargill M.A., Nakayama T., Ayabe T., Coleman M.L, & Bix M. (2019). Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway. PLoS ONE, 14(2), e0211244.

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