Anti mouse cd4 clone rm4 5

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The Anti-mouse CD4 (clone RM4-5) is a laboratory reagent used for the detection and analysis of mouse CD4-positive cells. It is a monoclonal antibody that specifically binds to the CD4 surface marker expressed on a subset of T lymphocytes in mice.

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6 protocols using anti mouse cd4 clone rm4 5

Comprehensive T Cell Profiling by Flow Cytometry

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T cells and their subsets were analyzed by differential expression of surface markers as well as by their intracellular cytokine profile. Briefly, cells were stained with different surface markers, such as anti-mouse CD3e (clone BM10-37, BD Biosciences), anti-mouse CD4 (clone RM4-5, BD Biosciences), anti-mouse CD8a (clone53-6.7, BD Biosciences) and anti-mouse CD25 (clone PC61, BD Biosciences). For analysis of the expression of intra cellular cytokines, cells were stimulated with 50 ng of PMA and 1 μg of ionomycin for 4 h in the presence of Golgi stop (BD Biosciences) to inhibit the secretion of the cytokines from the cells. Cells were then washed with staining buffer, fixed and permealized with BD cytofix and cytoperm^TMPlus (BD Biosciences) along with 0.2% Tween 20 for 20 minutes. Next, cells were stained with anti-mouse IL4 (clone 11B11, BD Biosciences), anti-mouse IFN-γ (clone XMG1.2, BD Biosciences), anti-mouse IL-17A (clone TC11-18H10) and anti-mouse Foxp3 (clone MF23, BD Biosciences). Brilliant stain buffer (BD Biosciences) and ultra comp beads (eBioscience, San Diego) were used for the dilution of the stains and compensation respectively. All the stained cells were analyzed by LSRII flow cytometer (BD Biosciences) and the data was evaluated by FlowJo software.

Bhattacharya S., Muhammad N., Steele R., Peng G, & Ray R.B. (2016). Immunomodulatory role of bitter melon extract in inhibition of head and neck squamous cell carcinoma growth. Oncotarget, 7(22), 33202-33209.

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Lung and Lymphoid Tissue Single-Cell Analysis

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Lung single-cell suspensions from vaccinated or Mtb-infected mice were generated as before4 (link). For LNs and spleens, organs were passed through a 70 μm cell strainer and then processed as for lungs4 (link). For flow cytometric analysis, cells were either stained immediately, or stimulated for 18 h in the presence of Ag85B_240–254 peptide, with GolgiStop (5 μl ml⁻¹; BD Biosciences) and Brefeldin A (1 μg ml⁻¹; BioLegend, San Diego, CA, USA) added for the last 5 h. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were collected using a BD FACS Jazz or a BD LSR Fortessa, and analysis was performed using FlowJo (Treestar, Ashland, OR, USA). Antibodies used include anti-mouse CD11c (clone HL3; BD Biosciences; dilution: 1/200), anti-mouse MHC-II (clone M5/114.15.2; Tonbo Biosciences, San Diego, CA, USA; dilution: 1/150), anti-mouse CD3 (clone 500A2; BD Biosciences; dilution: 1/200), anti-mouse CD4 (clone RM4-5; BD Biosciences, dilution: 1/200), anti-mouse CD44 (clone IM7; eBioscience, dilution: 1/400), anti-mouse IL-17 (clone TC11-18H10; BD Biosciences, dilution: 1/100), anti-mouse IFN-γ (XMG1.2; BD Biosciences; dilution: 1/100), anti-mouse Thy1.1 (clone OX-7; BD Biosciences; dilution: 1/100) and anti-mouse CD103 (clone 2E7, eBioscience; dilution: 1/200).

Griffiths K.L., Ahmed M., Das S., Gopal R., Horne W., Connell T.D., Moynihan K.D., Kolls J.K., Irvine D.J., Artyomov M.N., Rangel-Moreno J, & Khader S.A. (2016). Targeting dendritic cells to accelerate T-cell activation overcomes a bottleneck in tuberculosis vaccine efficacy. Nature Communications, 7, 13894.

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Murine Immune Cell Phenotyping

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Anti-IDH2 was from Abcam (# ab131263), anti-IDH1 was from Cell Signaling Technology (#8137S), and anti-HIF1α is from Novus biologicals (#NB100–105). Anti-mouse CD3 (clone 145–2C11, cat#16–0031), anti-mouse CD28 (clone 37.51, cat#16–0281), anti-mouse IFNγ (Clone XMG1.2,Cat#16–7311), anti-mouse IL-4(Clone 11B11, cat#16–7041), anti-mouse FOXP3 (clone FJK-16s, cat#17–5773) are from eBioscience. Anti-mouse CD4 (clone RM4–5, Cat#550954), anti-mouse CD25 (clone 7D4, Cat#558642), anti-mouse CD44 (Clone IM7, Cat#559250), anti-mouse CD62L (clone MEL-14, Cat#560507), anti-mouse IL-17 (clone TC11–18H10, Cat#559502), anti-mouse IFNγ (Clone XMG1.2, Cat#561040, for staining) and anti-CD90.1 (Thy1.1) (clone OX-7, cat#561406) were from BD Bioscience.

Xu T., Stewart K.M., Wang X., Liu K., Xie M., Ryu J.K., Li K., Ma T., Wang H., Ni L., Zhu S., Cao N., Zhu D., Zhang Y., Akassoglou K., Dong C., Driggers E.M, & Ding S. (2017). Metabolic control of TH17/iTreg balance by an epigenetic mechanism. Nature, 548(7666), 228-233.

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Maternal CD8+ T Cell Analysis

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Flow cytometry was used to analyze CD8⁺ T cells in the maternal blood and placenta. Isolated lymphocytes were stained with surface markers in 2% fetal bovine serum in PBS for 30 minutes at 4°C in the dark. Antibodies used were anti-mouse CD45⁺ (clone 30-F11, ThermoFischer Scientific, Waltham, MA, USA), anti-mouse CD3⁺ (clone 17A2, BioLegend, San Diego, CA, USA), anti-mouse CD4⁺ (clone RM4-5, BD, Franklin Lakes, NJ, USA) and anti-mouse CD8α or its isotope (clone 2.43, TONBO Biosciences, San Diego, CA, USA). For blocking experiments, cells were incubated with unlabeled anti-CD8⁺ antibodies (clones YTS169.9 and 53-6.72, Bio X Cell, West Lebanon, NH, USA) in fluorescence activated cell sorting (FACS) buffer for 20 minutes at 4°C in the dark prior to surface marker staining. Data were acquired on a BD FACSCalibur^TM flow cytometer with Cellquest software (BD Bioscience, San Jose, CA, USA) and analyzed with FlowJo version 10.1 (FlowJo, LLC, Ashland, OR, USA).

Lei J., Xie L., Zhao H., Gard C., Clemens J.L., McLane M.W., Feller M.C., Ozen M., Novak C., Alshehri W., Alhejaily N., Shabi Y., Rosenzweig J.M., Facciabene A, & Burd I. (2017). Maternal CD8+ T cell depletion alleviates intrauterine inflammation-induced perinatal brain injury. American journal of reproductive immunology (New York, N.Y. : 1989), 79(5), e12798.

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Quantifying IFN-γ Responses and T-cell Subsets

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The IFN-γ-secreting splenocytes were measured at 7, 17, and 45 dpi using a mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Z_EDIII (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB). To measure secreted cytokines, splenocytes (5 × 10⁵/well) were stimulated with Z_EDIII (1 μg/ml) for 48 h and the supernatant of splenocytes was collected. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate. To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (clone 145-2C11, BD Biosciences), and anti-mouse CD4 (clone RM4-5, BD Biosciences) or anti-mouse CD8 (clone 53–6.7, BD Biosciences) as described previously [27 (link)]. Cell phenotype was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

Shin M., Kim K., Lee H.J., Jung Y.J., Park J, & Hahn T.W. (2022). Vaccination with a Zika virus envelope domain III protein induces neutralizing antibodies and partial protection against Asian genotype in immunocompetent mice. Tropical Medicine and Health, 50, 91.

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Mouse Tumor Single-Cell Immune Profiling

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Preparation of single-cell suspension of mouse tumor tissues by mechanical grinding. All single-cell suspensions were incubated with rat anti-mouse CD16/CD32 blocking antibody (4 μg/ml) for 15 min after thorough filtration and precipitation, stained with fluorescein-conjugated antibodies, multiple washed with PBS, and resuspended in 7-AAD (exclude non-viable cells). For anti-mouse CD4 (Clone RM4-5, BD Biosciences), anti-mouse CD8 (clone 53-6.7, eBioscience), anti-mouse CD45 (clone 30-F11, Biolegend), anti-mouse CD25 (PC61.5, eBioscience), anti-mouse PD-L1 (clone 10 F.9G2, BioLegend) and anti-mouse CD31 (clone MEC 13.3, BD Biosciences) staining, after incubation for 1 h, cells were washed with PBS for three times (1500 rpm, 5 min each), then detected by flow cytometry (BD FACSCanto II). For FoxP3 staining, after incubation, cells were washed and fixed with 1 ml of fixation & permeabilization solution (BD Biosciences) for 30–60 min, and washed twice with Perm Wash (BD Biosciences). Intracellular staining with anti-FoxP3 antibody (clone FJK-16s, eBioscience) was performed for 1 h. The next steps are the same as described above.

Liu S., Qin T., Liu Z., Wang J., Jia Y., Feng Y., Gao Y, & Li K. (2020). anlotinib alters tumor immune microenvironment by downregulating PD-L1 expression on vascular endothelial cells. Cell Death & Disease, 11(5), 309.

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