Cd4 clone gk1

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD4 (clone GK1.5) is a laboratory research reagent used for the identification and analysis of T-helper cells. It binds to the CD4 cell surface marker, which is expressed on a subset of T lymphocytes. This product can be used in various immunological applications such as flow cytometry and cell sorting.

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CD4 (GK1.5, (30 citations) Anti-CD4 (clone GK1.5, (11 citations) CD4-FITC (clone GK1.5, (5 citations) Clone GK1.5 (4 citations) Rat anti-mouse CD4 (Clone GK1.5; (3 citations) Anti-CD4-PE (clone GK1.5; (2 citations) PerCP/Cy5.5-anti-CD4 (clone GK1.5), (2 citations) PE-Cyanine 7-conjugated anti-mouse CD4 (clone GK1.5, (2 citations) CD4 APC-eFluor 780 (clone GK1.5, (2 citations) FITC-conjugated anti-CD4 mAb (clone GK1.5; (2 citations)

15 protocols using cd4 clone gk1

Multiparameter Flow Cytometry Immunophenotyping

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CD45R/B220 (clone RA3-6B2, 1:100, eBioscience), CD3 (145-2c11, 1:100, eBioscience), CD45.2 (clone 104, 1:100, eBioscience), CD4 (clone GK1.5, 1:100, eBioscience), CD8 (clone 53-6.7, 1:100, eBioscience), CD19 (clone eBio1D3, 1:100, eBioscience), CD5 (clone 53-7.3, 1:100, eBioscience), CD21 (clone eBio4E3, 1:100, eBioscience), CD43 (clone eBioR2/60, 1:100, eBioscience), IgD (clone 11–26, 1:100, eBioscience), Gr1 (clone RB6-8C5, 1:100, eBioscience), IgM (clone 11f41, 1:100, eBioscience), GL-7 (clone GL-7, 1:100, eBioscience), Mac1 (clone M1/70, 1:100, eBioscience).

Souroullas G.P., Fedoriw Y., Staudt L.M, & Sharpless N.E. (2017). Lkb1 deletion in murine B lymphocytes promotes cell death and cancer. Experimental hematology, 51, 63-70.e1.

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Multiparametric Flow Cytometry Immunophenotyping

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Aliquots of 1 × 10⁶ cells were stained with different monoclonal antibodies according to standard protocols. The cells were analyzed on a FACSVerse cytometer (BD Biosciences, San Diego, CA, USA). Fluorescent antibodies of CD45 (clone 30-F-11), CD3ε (clone 145-2C11), CD4 (clone GK1.5), CD83 (clone Michel-19), CD11b (clone M1/70), ly6c(clone HK1.4), F4/80 (clone BM8), B220 (clone RA3-6B2), NK1.1 (clone PK136), MHC-II (clone M5/114.15.2), CD11c (clone N418), CD69 (clone H1.2F3), and Ki67 (clone B56) conjugated with the corresponding fluorescent dyes (eBioscience, San Diego, CA, USA) were used in the experiments.

Lin W., Zhou S., Feng M., Yu Y., Su Q, & Li X. (2021). Soluble CD83 Regulates Dendritic Cell–T Cell Immunological Synapse Formation by Disrupting Rab1a-Mediated F-Actin Rearrangement. Frontiers in Cell and Developmental Biology, 8, 605713.

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Comprehensive Immunophenotyping of T Cells

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The cells were stained with the following antibodies: CD8 (clone 53–6.7, eBioscience), CD4 (clone GK1.5, eBioscience), CD90.1 (clone OX-7, eBioscience), CD90.2 (clone 53–2.1, eBioscience), PD-1 (clone J43, Biolegend), KLRG1 (clone 2F1, Biolegend), p53 (clone pAb 240, Novus Biologicals) TNF (Clone MP6-XT22, Biolegend) and IFNγ (clone XMG1.2, Biolegend) at the appropriate dilution and with compatible fluorochromes. The lymphocyte gates in the samples were plotted directly to examine CD8 T cell populations depicted in the flow plots.

Kurup S.P., Moioffer S.J., Pewe L.L, & Harty J.T. (2020). P53 hinders CRISPR/Cas9 mediated targeted gene disruption in memory CD8 T cells in vivo. Journal of immunology (Baltimore, Md. : 1950), 205(8), 2222-2230.

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Isolation and Analysis of Intestinal Lymphocytes

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Caecum and proximal colon were excised and intra epithelial lymphocytes (IEL) and lamina propria lymphocytes (LILP) were prepared essentially as described [52 (link)] with slight ±modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) and processed with ebioscience fix/perm buffer as per manufacturer’s instructions before labelling with antibodies specific for CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience) and T-bet (clone TWAJ; eBioscience). All samples were analysed on a FACS LSRII.

Houlden A., Hayes K.S., Bancroft A.J., Worthington J.J., Wang P., Grencis R.K, & Roberts I.S. (2015). Chronic Trichuris muris Infection in C57BL/6 Mice Causes Significant Changes in Host Microbiota and Metabolome: Effects Reversed by Pathogen Clearance. PLoS ONE, 10(5), e0125945.

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Intracellular Cytokine Staining of Lung Cells

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Cells from lungs digested using the collagenase single-cell suspension protocol were resuspended in RPMI-1640 media containing 10% fetal bovine serum (FBS), penicillin (100U/mL)/streptomycin (100 μg/mL), 1mM sodium pyruvate, 0.2μM 2-mercaptoethanol, 2mM L-glutamine, 0.1mM non-essential amino acids, and 10mM HEPES buffer and cultured at 3×10⁶ cells/well in 6-well cell culture-treated plates (Corning, Corning, NY), with cells from each mouse incubated separately in their own well. PMA (LC Laboratories, Woburn, MA) at a final concentration of 100 ng/mL and ionomycin (Sigma, St. Louis, MO) at a final concentration of 1500 ng/mL were used to stimulate the cells for 1 hour at 37°C with 5% CO₂ before GolgiStop (BD Biosciences, San Jose, CA) was added; cells were cultured an additional 4 hours. Intracellular cytokine staining (ICS) was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences, San Jose, CA) and antibodies to label the following cell surface markers and cytokines were used: CD45 (clone 30-F11, Biolegend, San Diego, CA), CD3ε (clone 145-2C11, Biolegend), CD4 (clone GK1.5, eBioscience, San Diego, CA), IL-17A (clone eBio17B7, eBioscience), and IFN-γ (clone XMG1.2, eBioscience). Stained cells were analyzed using a LSRII (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Smith N.M., Wasserman G.A., Coleman F.T., Hilliard K.L., Yamamoto K., Lipsitz E., Malley R., Dooms H., Jones M.R., Quinton L.J, & Mizgerd J.P. (2017). Regionally Compartmentalized Resident Memory T Cells Mediate Naturally Acquired Protection Against Pneumococcal Pneumonia. Mucosal immunology, 11(1), 220-235.

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Cytokine profiling of obese WT and IFNAR-/- mice

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eWAT and liver was isolated from obese WT and IFNAR^−/− mice. eWAT-isolated SVF cells and liver immune cells were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (1 μg/ml; Calbiochem). Briefly, cells were stained with Live/dead stain (Zombie UV Dye; 1:250; Biolegend), B220 (clone RA3-6B2; 1:100; Biolegend), CD45 (clone 104; 1:500), CD11b (clone M1/70; 1:100), F4/80 (clone BM8; 1:100), Gr1 (clone RB6-8C5; 1:100), CD4 (clone GK1.5; 1:50), CD8 (clone 53-6.7; 1:100), IFNγ (clone XMG1.2; 1:100) TNF (clone MP6-XT22; 1:100), and IL-6 clone (MP5-20F3; 1:100) (all ebioscience). Data were collected using a LSR Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Chan C.C., Damen M.S., Moreno-Fernandez M.E., Stankiewicz T.E., Cappelletti M., Alarcon P.C., Oates J.R., Doll J.R., Mukherjee R., Chen X., Karns R., Weirauch M.T., Helmrath M.A., Inge T.H, & Divanovic S. (2020). Type I interferon sensing unlocks dormant adipocyte inflammatory potential. Nature Communications, 11, 2745.

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Flow Cytometric Analysis of Immune Cells

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For flow cytometric analysis up to 1 × 10⁶ cells per tube were stained with surface antibodies at 1:1000 (CD45 (clone 104, eBioscience), CD3 (clone 17A2, eBioscience), CD4 (clone GK1.5, eBioscience)) for 20 min at RT. Cells were then fixed with 100 μl Fix&Perm Reagent A (Invitrogen) for 15 min at RT and then stained with 1 μl of intracellular antibodies (FoxP3 (clone FJK-16 s, eBioscience)) in 100 μl Fix&Perm Reagent B (Invitrogen) overnight at 4 °C. Fixed and permeabilized unstained cells were used as a negative control, single color surface stained cells or UltraComp beads (eBioscience) were used for compensation and Fluorescence Minus One (FMO), meaning cells that were identically stained as samples, but were lacking one staining antibody at a time, were used to control for multiple fluorochrome staining particularly for intracellular targets. Cells and controls were analyzed using a BD FACS Canto II cytometer and data were subsequently analyzed using FlowJo software (Treestar Inc.).

Dittmer M., Young A., O’Hagan T., Eleftheriadis G., Bankhead P., Dombrowski Y., Medina R.J, & Fitzgerald D.C. (2018). Characterization of a murine mixed neuron-glia model and cellular responses to regulatory T cell-derived factors. Molecular Brain, 11, 25.

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Immune Cell Analysis in Virus-Infected Mice

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Mice infected with virus for 10 days were anesthetized and perfused. Mouse spleens were harvested and homogenized. Splenocytes were treated twice with buffer to lyse red blood cells and washed. In addition, leukocytes in the brain were separated by discontinuous Percoll gradient and washed. Cells were stained with FITC- or PE-conjugated control antibodies or monoclonal antibodies against CD4 (clone GK 1.5, eBioscience), CD8a (clone 53–6.7, eBioscience), CD19 (clone 6D5, eBioscience), CD69 (clone H1.2F3, eBioscience), or CD138 (clone 281–2, BD Biosciences). The stained cells were analyzed by a FACSCalibur (BD Biosciences) and data were analyzed by WinMDI software.

Wang L.C., Yao H.W., Chang C.F., Wang S.W., Wang S.M, & Chen S.H. (2017). Suppression of interleukin-6 increases enterovirus A71 lethality in mice. Journal of Biomedical Science, 24, 94.

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Immunohistochemistry of Murine Brain Sections

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Sections (10 µm) from snap-frozen mouse brains fixed with acetone were blocked for 60 min (DAKO REAL Peroxidase-blocking solution, DAKO serum-free protein block) and incubated for 60 min with antibodies against adenovirus (Millipore), CD8α (clone 53–6.7, eBioscience), or CD4 (clone GK1.5, eBioscience) followed by the secondary antibodies rabbit anti-goat IgG or goat anti-rat IgG (Invitrogen). Sections were stained with DAB chromogen (DAKO) and hematoxylin (Sigma-Aldrich) and mounted with Pertex (Histolab). Sections were photographed on an Axio Observer D1 microscope with 40× magnification (Zeiss).

Belcaid Z., Berrevoets C., Choi J., van Beelen E., Stavrakaki E., Pierson T., Kloezeman J., Routkevitch D., van der Kaaij M., van der Ploeg A., Mathios D., Sleijfer S., Dirven C., Lim M., Debets R, & Lamfers M.L. (2020). Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy. Neuro-oncology Advances, 2(1), vdaa011.

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Isolation and Analysis of Mouse Hematopoietic Stem Cells

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Cells were analyzed using LRSII (BD, Pharmingen) and sorted using FACS-ARIA II (BD, Pharmingen). The following antibodies were used: anti-CD45 FITC (clone 30-F11 Biolegend), anti-CD31 FITC (clone MEC13.3 Biolegend), anti-Ter119 FITC (clone TER-119 Biolegend), anti-Sca1 Pacific Blue (clone D7 Biolegend), anti-CD51 PE (clone RMV-7 Biolegend), bio-Lineage panel antibodies [CD4 (clone GK1.5 eBioscience), CD8 (clone 53-6.7 eBioscience), CD3 (clone 145-2C11 eBioscience), Ter119 (clone TER-119 eBioscience), CD11b (clone M1/70 eBioscience), Gr1 (clone RB6-8C5 eBioscience), NK1.1 (clone PK136 eBioscience), B220 (clone RA3-6B2 eBioscience)], anti-ckit APC (clone 2B8 eBioscience), anti-Sca1 PE (clone D7 eBioscience), anti-CD34 FITC (clone RAM-34 eBioscience), anti-SLAM (CD150) PerCP cy5.5 (clone TEC15-12F12.2 Biolegend), anti-Cd11b APC (clone M1/70 Biolegend), and anti-Gr1 PE (clone RB6-8C5 Biolegend).

Guarnerio J., Mendez L.M., Asada N., Menon A.V., Fung J., Berry K., Frenette P.S., Ito K, & Pandolfi P.P. (2018). A non-cell-autonomous role for Pml in the maintenance of leukemia from the niche. Nature Communications, 9, 66.

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