Clone rm4 5

Manufactured by BioLegend

Sourced in United States

Clone RM4-5 is a mouse monoclonal antibody that recognizes the CD4 surface antigen. It can be used for the identification and enumeration of CD4+ T cells in flow cytometric analysis.

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7 protocols using clone rm4 5

Comprehensive Immunophenotyping by Flow Cytometry

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Flow cytometry analysis was performed using a Becton-Dickinson LSR Fortessa 20X or CANTO II. We used the following antibodies: CD4 (clone RM4-5, BV510 or FITC, Biolegend), CD25 (3C7, PE, Biolegend or PC61, BV605, Biolegend), Siglec-F (LOU, BV421, BD Biosciences), Foxp3 (FJK-16s, APC, eBioscience), Helios (22F6, Pacific blue, Biolegend), CD11c (N418, APC, Biolegend), MHCII (M5/114.15.2, BV510, Biolegend), CD11b (M1/70, PercP, Biolegend), Ly6G (1A8, PE/Cy7, Biolegend), Ly6C (HK1.4, PE, Biolegend), IL-13 (eBio13A, PE, eBioscience), IL-17A (TC11-18H10.1, PercP/Cy5.5 or AlexaFluor700, Biolegend), IFNγ (4S.B3, BV421 or XMG1.2, APC/Cy7, Biolegend), CD45 (30-F11, Alexa Fluor700 or APC, Biolegend), fixable viability dye (Zombie Aqua, Biolegend), and fixable viability dye (eFluor780, eBioscience).

Dembele M., Tao S., Massoud A.H., Miah S.M., Lelias S., De Groot A.S, & Mazer B.D. (2021). Tregitopes Improve Asthma by Promoting Highly Suppressive and Antigen-Specific Tregs. Frontiers in Immunology, 12, 634509.

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Multiparametric Phenotyping of Immune Cells

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Single cell suspensions from the ear were obtained as described above. For analysis of surface markers and intracellular cytokines, some cells were incubated for 4 h with 10 µg/mL of brefeldin A, 50 ng/mL of PMA and 500 ng/mL ionomycin (Sigma-Aldrich). Before staining, cells were incubated with anti-mouse CD16/CD32 mouse Fc block (Thermo Fisher Scientific, RRID:AB_467135) and 10% rat-IgG in PBS containing 0.1% BSA. Cells were stained for dead cells with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Molecular Probes) and surface markers (CD4 [BioLegend, clone RM4-5, RRID:AB_11219790], CD8β [BioLegend, clone YTS156.7.7, RRID:AB_2260149], TCRγδ [BD Biosciences, clone GL3, RRID: AB_2661844], CD45 [Thermo Fisher Scientific, clone 30-F11, RRID:AB_493714], Ly6G [Thermo Fisher Scientific, clone 1A8-Ly6g, RRID:AB_2637123], CD11b [BioLegend, clone M1/70, RRID:AB_11125575]) followed by fixation with 2% of formaldehyde and permeablization with 0.2% saponin/PBS. Intracellular cytokine staining was performed for pro-IL-1β (Thermo Fisher Science, clone NJTEN3, RRID:AB_10670739), IL-17 (Thermo Fisher Scientific, clone eBio17B7, RRID:AB_763579), and IFN-γ (Thermo Fisher Scientific, clone XMG1.2, RRID:AB_1257211). The data were collected using LSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star).

Gimblet C., Meisel J.S., Loesche M.A., Cole S.D., Horwinski J., Novais F.O., Misic A.M., Bradley C.W., Beiting D.P., Rankin S.C., Carvalho L.P., Carvalho E.M., Phillip S, & Grice E.A. (2017). Cutaneous leishmaniasis induces a transmissible dysbiotic skin microbiota that promotes skin inflammation. Cell host & microbe, 22(1), 13-24.e4.

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Multicolor Flow Cytometry Phenotyping

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Immunophenotyping of cells was performed by flow cytometry using multicolor fluorochrome-conjugated antibodies. Identification of cell surface markers was performed using antibodies purchased from BD Biosciences (San Jose, CA, USA; anti-CD11c FITC; clone HL3, anti-MHCII PE; clone 2G9, anti-CD86 APC; clone 2331 [FUN-1], anti-CD4 PerCP-Cy; clone RM4–5, anti-CD11b PerCP-Cy; clone M1/70, anti-F4/80 BV421; clone T45-2342) or BioLegend (San Diego, CA, USA; anti-CD80 PE/Cy7; clone 16-10A1) and used at a dilution of 1:100–1:500 unless stated otherwise. After surface-staining, anti-IFN-γ FITC (clone B27) and anti-IL-17 PE (clone TC11-18H10) (BD Biosciences) were used for intracellular staining using a fixation and permeabilization kit (BD Biosciences). Flow cytometry data were acquired on a BD LSR Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Ye B.J., Lee H.H., Yoo E.J., Lee C.Y., Lee J.H., Kang H.J., Jeong G.W., Park H., Lee-Kwon W., Choi S.Y, & Kwon H.M. (2020). TonEBP in dendritic cells mediates pro-inflammatory maturation and Th1/Th17 responses. Cell Death & Disease, 11(6), 421.

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Phenotypic Analysis of Tumor-Infiltrating Immune Cells

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The filtered tumor tissue cells were blocked with an anti-CD16/32 antibody (catalog 101319; 1 μg per 106 cells in 100 μL dilution buffer; BioLegend) and stained with indicated surface antibodies. Dead cells were marked using a Live/Dead Fixable Aqua dye (catalog L34965, Thermo Fisher Scientific). Fluorochrome-conjugated or biotinylated antibodies and their source, dilution information, and manufacturer are as follows: PerCP-Cy5.5–anti–mouse CD45 (1:200; clone 30-F11, catalog 103131, BioLegend), PE–Dazzle anti–mouse CD3ε (1:200; clone 145-2C11, catalog 100347, BioLegend), APC-Cy7–anti–mouse CD4 (1:200; clone RM4-5, catalog 100526, BioLegend), Alexa Fluor 700–anti–mouse CD8a (1:200; clone 53-6.7, catalog 100730, BioLegend), PE–anti–mouse IFN-γ (1:200; clone XMG1.2, catalog 505808, BioLegend), PE-Cyanine7–anti–mouse granzyme B (1:200; clone NGZB, catalog 25-8898-82, Invitrogen). Intracellular antibodies were added after fixation (catalog 420801, BioLegend) and permeabilization (catalog 421002, BioLegend), according to the manufacturer’s instructions. A Beckman Coulter CytoFLEX was used for our analysis, and FlowJo (version 10.8.1, Tree Star) was used for data analysis. Supplemental Figure 1E details the flow cytometry gating strategy.

Li K., Liu W., Yu H., Chen J., Tang W., Wang J., Qi M., Sun Y., Xu X., Zhang J., Li X., Guo W., Li X., Song S, & Tang S. (2024). 68Ga-FAPI PET imaging monitors response to combined TGF-βR inhibition and immunotherapy in metastatic colorectal cancer. The Journal of Clinical Investigation, 134(4), e170490.

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T cell phenotyping by flow cytometry

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GLN and MLN were manually dissociated into single cell suspensions. A total of 1–2 × 10⁶ cells were incubated with Zombie violet (BioLegend, San Diego, CA) and Fc block (anti CD16/CD32, Clone 93, BioLegend, San Diego, CA). T cell surface markers, CD4 and CD3, were detected using CD4-PerCP (1:200; Clone RM4-5; BioLegend, San Diego, CA) and CD3-APC/Cy7 (1:100; Clone 17A2; BioLegend, San Diego, CA). Cells were fixed and permeabilized using Fix/Perm solution (BioLegend, San Diego, CA). Transcription factors were detected using the following antibodies: T-bet-FITC (1:100; Clone 4B10; BioLegend, San Diego, CA), RORγT-APC (1:100; Clone B2D; Thermo Fisher Scientific, Waltham, MA), or FOXP3-PE (1:100; Clone FJK-16s; Thermo Fisher Scientific, Waltham, MA). Samples were analyzed on a BD LSR II Flow Cytometer.

Lunger C., Shen Z., Holcombe H., Mannion A.J., Dzink-Fox J., Kurnick S., Feng Y., Muthupalani S., Carrasco S.E., Wilson K.T., Peek R.M., Piazuelo M.B., Morgan D.R., Armijo A.L., Mammoliti M., Wang T.C, & Fox J.G. (2023). Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer. Microbiology Spectrum, 12(1), e03450-23.

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Immunofluorescent Staining of CD4+ T Cells

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Liver tissue sections were prepared on slides, rehydrated in water, washed with PBS, 0.05% Tween20, and permeabilized for 15 min with PBS containing 0.1% Triton X-100. Slides were then blocked with blocking buffer (PBS with 1% BSA, 0.1% fish gelatin, 0.1% Triton-X-100, 0.05% Tween 20, and 5% animal serum, corresponding to the host species of the secondary antibody). Slides were stained with rat anti-mouse CD4 (1 μg/ml, Clone RM4-5, Biolegend) in blocking buffer overnight at 4°C. Slides were then washed three times in wash buffer and stained with Alexa Fluor–594 conjugated goat anti-rat secondary antibody (0.2 μg/ml, Life Technologies) and solubilized in blocking buffer for 2 hours at room temperature. After being washed three times, the cells were stained with Hoechst 33342 nuclear dye (0.2 μg/ml) for 10 min at room temperature. Finally, the slides were washed twice and coverslips (FischerBrand, 22 mm #1) were mounted over VectaMount mounting medium (Vector Laboratories). Mounted samples were cured overnight at room temperature and imaged within 24 hours. All images were captured on a Zeiss AxioObserver microscope with a Zeiss 40X (1.3 NA) objective lens and Hamamatsu CMOS OrcaFlash 4.0 sCMOS camera. Images were then processed with NIH ImageJ software.

Mendu S.K., Stremska M.E., Schappe M.S., Moser E.K., Krupa J.K., Rogers J.S., Stipes E.J., Parker C.A., Braciale T.J., Perry J.S, & Desai B.N. (2020). Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling. Science signaling, 13(661), eabb0619.

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Isolation and Activation of Murine Splenocytes

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The spleen was harvested from a laboratory BALB/c mouse. After grinding and passing through the 200 meshes strainer, splenocytes were resuspended in RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamate, penicillin (100 U/mL), streptomycin (100 μg/mL), 2 μg/mL anti- mouse CD3 (Cat 100301, Clone 145-2C11, BioLegend), 1 μg/mL anti- mouse CD28 (Cat 102101, Clone 37.51, Biolegend), and 20 ng/mL recombinant mouse IL-2 (PeproTech). A total of 1 × 10⁶ splenocytes per well were seeded into a 12-well plate, treated with 100 nM NP-IDO-APT, NP-Scr-APT, or NPs, and cultured for 6 days. For T cell function measurement, cells were stimulated with 100 ng/mL PMA, 500 ng/mL ionomycin, and protein transport inhibitor cocktail (eBioscience) for 5 h before harvest. Afterwards, cells were incubated with anti-CD45-PEcy7(Cat 103113, Clone 30-F11, Biolegend), anti-CD8-FITC (Cat 100705, Clone 53-6.7, Biolegend) for surface staining and incubated with anti-IFN-γ-APC (Cat 505809, Clone XMG1.2, Biolegend) or anti-TNF-α-PE (Cat 506305, Clone MP6-XT22, Biolegend) after fixation and permeabilization. For T_reg cells analysis, cells were collected and stained with anti-CD4-PerCP (Cat 100537, Clone RM4-5, Biolegend), anti-CD25-PE (Cat 102007, Clone PC61, Biolegend), and anti-Foxp3-APC (Cat 77-5775-40, Clone FJK-16s, eBioscience). Subsequently, cells were measured by flow cytometry.

Zhu Z., Yang Z., Zhu C., Hu Z., Jiang Z., Gong J., Yuan Y., Chen X., Jin Y, & Yin Y. (2023). Development of a DNA aptamer targeting IDO1 with anti-tumor effects. iScience, 26(8), 107367.

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