Cd4 clone rm4 5

Manufactured by BD

Sourced in United States

The CD4 (clone RM4-5) is a lab equipment product that is used to identify and analyze CD4+ T cells. It is a monoclonal antibody that binds specifically to the CD4 surface antigen expressed on helper T cells.

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Lab products found in correlation

Cd8a clone 53 6, by BD (5 mentions) Cd11c clone hl3, by BD (3 mentions) Cd8 clone 53 6, by BD (3 mentions) Facscanto 2, by BD (2 mentions) Facscalibur flow cytometer, by BD (2 mentions) Gr1 clone rb6 8c5, by BD (2 mentions) Cd11c clone n418, by BD (2 mentions) Nk1.1 clone pk136, by BD (2 mentions) Brefeldin a golgiplug, by BD (1 mentions) L glutamine, by Merck Group (1 mentions) Cytofix perm kit, by BD (1 mentions) Cd45.1 clone a20, by BD (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Cd127 clone a7r34, by BioLegend (1 mentions) Anti cd4 gk1, by BioXCell (1 mentions) Anti cd8 53.5.8, by BioXCell (1 mentions) Cd3 clone 17a2, by BD (1 mentions) Lsrii ow cytometer, by BD (1 mentions) Apotome, by Zeiss (1 mentions) Hoechst, by Merck Group (1 mentions)

Close spelling variants of this product

RM4-5 (42 citations) CD4 (RM4-5, (35 citations) Anti-CD4 (clone RM4-5, (13 citations) Anti-mouse CD4 (clone RM4-5, (6 citations) Clone RM4-5 (4 citations) FITC-conjugated anti-CD4 (RM4–5 clone; (3 citations) Anti-CD4 PerCP-Cy5.5 (clone RM4-5; (3 citations) Anti-CD4 PE (clone RM4-5, (3 citations) Anti-mouse CD4-V450 (clone RM4-5; (2 citations) CD4 (V450 clone RM4-5; (2 citations)

14 protocols using cd4 clone rm4 5

Multiparametric Flow Cytometry of Immune Cells

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Cell cultures were performed in complete culture medium consisting of RPMI-1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine (Sigma, St. Louis, MO), 50 μg/ml gentamicin (PAA, Linz, Austria), and 50 μM beta-mercaptoethanol (Sigma).
Phenotypical analyses were performed by flow cytometry with mAb against CD4 (clone RM4-5), CD45.1 (clone A20), MHC class II (anti-I-A/I-E^diverse, clone 2G9), CD11c (clone HL3), CD8α (clone Ly-2), CD45 (clone 30-F11), CD103 (clone M290), EpCAM/CD326 (clone G8.8), CD19 (clone 1D3), NK1.1 (clone PK136), IL-12p40 (clone C15.6) (all from BD-Pharmingen, San Diego, CA), CD127 (clone A7R34, Biolegend, San Diego, CA), CD70 (clone FR70, Biolegend), and Langerin/CD207 mAb (clone 929F3; Dendritics, Lyon, France). When possible, viable cells were determined by exclusion of 7-AAD-positive dead cells (BD-Pharmingen). IL-12p40 and IL-10 stainings were performed on total lymph node cell suspension (10⁶ cells/ml) incubated for 3 h in 1 μg/ml Brefeldin A (Golgiplug, BD-Pharmingen). To stain for Langerin or intracellular cytokines, permeabilization was performed with Cytofix/perm kit (BD-Pharmingen).

Flacher V., Tripp C.H., Mairhofer D.G., Steinman R.M., Stoitzner P., Idoyaga J, & Romani N. (2014). Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance. EMBO Molecular Medicine, 6(9), 1191-1204.

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T Cell Depletion in BALB/c Mice

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BALB/c mice were depleted of CD4⁺, CD8⁺, or a combination of CD4⁺ and CD8⁺ T cells. Depleting antibodies (anti-CD4⁺ [GK1.5] and anti-CD8⁺ [53.5.8] antibodies; Bio X Cell, Lebanon, NH) were administered at 250 μg/mouse by intraperitoneal injection on days −2, −1, 4, and 8 relative to the time of challenge on day 0. Control mice received a dose of rat Ig control (Sigma-Aldrich) in parallel. Depletions were verified on days 1, 9, and 16 by staining spleen cells derived from unchallenged vaccinated mice with CD3 (clone 17A2 labeled with V450), CD4 (clone RM4-5 labeled with V500), and CD8 (clone 53.6.7 labeled with PerCp-Cy5.5) antibodies (BD Biosciences).

Low L.M., Ssemaganda A., Liu X.Q., Ho M.F., Ozberk V., Fink J., Sundac L., Alcorn K., Morrison A., O’Callaghan K., Gerrard J., Stanisic D.I, & Good M.F. (2018). Controlled Infection Immunization Using Delayed Death Drug Treatment Elicits Protective Immune Responses to Blood-Stage Malaria Parasites. Infection and Immunity, 87(1), e00587-18.

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Multicolor Flow Cytometry of Transgenic T-Cells

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All staining procedures were performed on ice. Cells were incubated with anti-FcR (clone 24G2, lab grown) antibody for 20 minutes on ice, then stained with antibodies. The presence of OVA-transgenic T-cells was determined by staining with fluorescently labeled antibodies: CD4 (clone RM4-5; BD) in combination with the anti-DO11.10 TCR antibody clone KJ1-26 (lab grown). The cells were analyzed on a LSRII ﬂow cytometer (BD) or FACS Canto II (BD) in association with FlowJo software (Treestar).

Mahapatra S., Albrecht M., Behrens B., Jirmo A., Behrens G., Hartwig C., Neumann D., Raap U., Bähre H., Herrick C, & Dittrich A.M. (2014). Delineating the Role of Histamine-1- and -4-Receptors in a Mouse Model of Th2-Dependent Antigen-Specific Skin Inflammation. PLoS ONE, 9(2), e87296.

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Quantifying Tumor-Infiltrating Lymphocytes Using Immunofluorescence

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8-μm sections of cryo-conserved organs were attached on Superfrost slides, dried overnight at room temperature and fixed in 4% para-formaldehyde (PFA) for 10 min at room temperature in the dark. Sections were washed 3 times with PBS and blocked using PBS supplemented with 1% BSA, 5% mouse serum, 5% rat serum and 0.02% Nonident for 1 h at room temperature in the dark. Fluorescent labelled antibodies (FoxP3, clone FJK-16 s, eBioscience; CD8, clone 53-6.7, BD; CD4, clone RM4-5, BD) were diluted in staining buffer (PBS supplemented with 1% BSA, 5% mouse serum and 0.02% Nonident) and sections were stained overnight at 4 °C. After washing twice with washing buffer (PBS supplemented with 1% BSA and 0. 02% Nonident) and once with PBS, slides were stained for 3 min with Hoechst (Sigma), washed 3 times with PBS, once with distilled water and mounted using Mounting Medium Flouromount G (eBioscience). Immunofluorescence images were acquired using an epifluorescence microscope (ApoTome, Zeiss). Tumour, CD4, CD8 and FoxP3 stained areas were quantified within manually pre-defined tumour regions via computerized image analysis software (Tissue Studio 3.6.1., Definiens). The proportion of marker positive cells in comparison to DAPI positive cells was calculated.

Kreiter S., Vormehr M., van de Roemer N., Diken M., Löwer M., Diekmann J., Boegel S., Schrörs B., Vascotto F., Castle J.C., Tadmor A.D., Schoenberger S.P., Huber C., Türeci Ö, & Sahin U. (2015). Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature, 520(7549), 692-696.

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Multiparametric Flow Cytometry Analysis

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Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated monoclonal antibodies specific for mouse CD71 (clone C2), CD117 (clone 2B8), Sca-1 (clone D7), TER-119 (clone Ter-119), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), F4/80 (clone BM8), CD11c (clone N418), CD317 (clone eBio927), CD4 (clone RM4-5), CD8a (clone 53-6.7), CD3e (clone 145-2C11), B220 (clone RA3-6B2), CD19 (clone 1D3), CD41 (clone MWReg30), CD42d (clone 1C2), NK-1.1 (clone PK136), and FceR1 (clone MAR-1) (from BD Biosciences and eBioscience). Stained cells were washed and analyzed by 4-color flow cytometry on a FACSCalibur flow cytometer (BD Biosciences) at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center. Data were collected by using the Cell Quest software (BD Biosciences) and analyzed by using the Summit software (Dako Colorado, Inc.). At least 15,000 total events were analyzed. Dead cells were excluded according to staining with 7-amino-actinomycin D. For apoptosis analysis, the cells were stained with FITC-Annexin V and propidium iodide.

Zhao W., Zou K., Farasyn T., Ho W.T, & Zhao Z.J. (2014). Generation and Characterization of a JAK2V617F-Containing Erythroleukemia Cell Line. PLoS ONE, 9(7), e99017.

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Skin Histology and Immunohistochemistry

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Dorsal skin was collected and processed as described previously for histology and immunohistochemistry^{44 (link)}. Formalin-fixed, paraffin-embedded skin was sectioned and stained with haematoxylin and eosin (H&E) using standard techniques as described^{44 (link)}. Fresh frozen skin was sectioned and stained, as previously published^{44 (link), 49 (link)}, using antibodies targeting the following proteins: CD4 (clone RM4-5, Cat.#550280), CD8a (clone 53-6.7, Cat.#550281), CD11c (clone HL3, Cat.#550283; all BD Pharmingen; San Jose, CA) and F4/80 (clone BM8, Cat.#14-4801, eBioscience; San Diego, CA).
Epidermal thickness (acanthosis) measurements and immune cell quantification in dorsal skin sections was completed using microscopic images collected from the stained sections using interactive image analyses approaches as described previously^{44 (link)}.

Arbiser J.L., Nowak R., Michaels K., Skabytska Y., Biedermann T., Lewis M.J., Bonner M.Y., Rao S., Gilbert L.C., Yusuf N., Karlsson I., Fritz Y, & Ward N.L. (2017). Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis. Scientific Reports, 7, 11198.

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Immunohistochemistry and Flow Cytometry Protocol

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Five micron paraffin embedded tissue sections were processed for immuno-histochemistry as previously described (Sur et al., 2012b (link)). Rabbit polyclonal anti-Myc (Santa Cruz, sc-764; RRID:AB_631276) (1:500), Rabbit monoclonal anti Ki-67 (abcam, ab16667; RRID:AB_302459) (1:200), Goat polyclonal anti-Vimentin (Santa Cruz, sc-7557; RRID:AB_793998) (1:500), biotinylated goat anti-Rabbit IgG (Vector Laboratories, BA1000; RRID:AB_2313606) and biotinylated rabbit anti-Goat IgG (Vector Laboratories, BA5000; RRID:AB_2336126) (1:350) antibodies were used. For flow cytometry, single cell suspensions of spleen and bone-marrow and cells from peripheral blood were stained with Fc-block (CD16/CD32 clone 93, Biolegend, 101302, RRID:AB_312801) and subsequently with CD19 (clone 1D3, BD Biosciences, RRID:AB_11154223), TER119 (clone TER119, Biolegend 116210, RRID:AB_313711), CD3ε (clone 145–2 C11, Biolegend 100308, RRID:AB_312673), NK1.1(clone PK136, Biolegend, 108716, RRID:AB_493590), GR1/LY6G (clone RB6-8C5, Biolegend, 108410, RRID:AB_313375), CD4 (clone RM4-5, BD Biosciences, 563747) and CD8a (clone 53–6.7, BD Biosciences, 563332). Dead cells were visualized using Propidium iodide. Samples were analyzed using a BD LSRFortessa instrument.

Dave K., Sur I., Yan J., Zhang J., Kaasinen E., Zhong F., Blaas L., Li X., Kharazi S., Gustafsson C., De Paepe A., Månsson R, & Taipale J. (2017). Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth. eLife, 6, e23382.

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Lung Leukocyte Profiling in Mice

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In mouse models, BAL was performed by cannulation through the trachea and lavage with 1.5 ml sterile PBS. Cells were pelleted by centrifugation, resuspended in ACK buffer to lyse red cells, washed in PBS and resuspended in RPMI medium with 10% FBS. Cells were stained with Quik Diff (Reagena) for differential counting. For flow cytometry analysis, lung leukocytes were obtained from BAL and red cells were lysed with ACK buffer. BAL cells were stained with Live/Dead fixable dead cell stain (Life Technologies, Carlsbad, CA), incubated with anti-mouse CD16/CD32 (FC block; BD Biosciences) and subsequently with directly fluorochrome-conjugated monoclonal antibodies specific for CD3ε (clone 500 A; 2 1 µg/ml, BD Biosciences), CD69 (clone H1.2F3 1 µg/ml, BD Biosciences), CD4 (clone RM4-5 0.25 µg/ml; BD Biosciences), CD8a (clone 53–6.7; 0.5 µg/ml BD Biosciences) and NK1.1 (clone PK136;1 µg/ml, BD Biosciences). Data were acquired on an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (version 10.0.6; Tree Star, Ashland, USA). Representative gating strategies used for analysis of cell surface staining are shown in Supplementary Fig. 8.

Singanayagam A., Glanville N., Girkin J.L., Ching Y.M., Marcellini A., Porter J.D., Toussaint M., Walton R.P., Finney L.J., Aniscenko J., Zhu J., Trujillo-Torralbo M.B., Calderazzo M.A., Grainge C., Loo S.L., Veerati P.C., Pathinayake P.S., Nichol K.S., Reid A.T., James P.L., Solari R., Wark P.A., Knight D.A., Moffatt M.F., Cookson W.O., Edwards M.R., Mallia P., Bartlett N.W, & Johnston S.L. (2018). Corticosteroid suppression of antiviral immunity increases bacterial loads and mucus production in COPD exacerbations. Nature Communications, 9, 2229.

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T-cell Characterization by FACS

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T-cell analyses were performed by fluorescence-activated cell sorting (FACS) using an LSRII or a FACS Calibur flow cytometer and the cell quest analysis program (BD Bioscience, Heidelberg, Germany). T-cell surface markers were directly stained with fluorescence-labeled mAbs against CD4 (clone RM4-5; BD Biosciences), CD8 (53-6.7, eBioscience, San Diego, USA), CD44 (IM7; BD Biosciences), CD62L (MEL-14; eBioscience) and CD69 (H1.2F3; BD Bioscience). Ki67 (SolA15; eBioscience), CTLA-4 (UC10-4B9; BioLegend, or HB304) and the transcription factor FoxP3 (FJK-16s; eBioscience) were stained intracellularly.

Deppisch N., Ruf P., Eißler N., Lindhofer H, & Mocikat R. (2016). Potent CD4+ T cell-associated antitumor memory responses induced by trifunctional bispecific antibodies in combination with immune checkpoint inhibition. Oncotarget, 8(3), 4520-4529.

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Lymphocyte Depletion Analysis by Flow Cytometry

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Lymphocyte depletion was confirmed by staining splenocytes with antibodies to TCR β chain (clone H57-597, BD Pharmingen), CD8 (clone 53-6.7, BD Pharmingen), and CD4 (clone RM4-5, BD Pharmingen). Cells were preincubated with anti-FcγR mAb 2.4G2 to block nonspecific binding before staining. Stained samples were analyzed by flow cytometry with a CyAn ADP flow cytometer (Beckman Coulter, Inc.) and FlowJo (Tree Start, Inc.) or Summit (Beckman Coulter, Inc.) software.

Molloy C.T., Andonian J.S., Seltzer H.M., Procario M.C., Watson ME J.r, & Weinberg J.B. (2017). Contributions of CD8 T Cells to the Pathogenesis of Mouse Adenovirus Type 1 Respiratory Infection. Virology, 507, 64-74.

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