Cd4 apc

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The CD4-APC is a cell surface marker used for the identification and enumeration of CD4+ T cells in flow cytometry analysis. It is a fluorescently labeled antibody that binds specifically to the CD4 antigen expressed on the surface of T helper cells. The APC (Allophycocyanin) fluorochrome attached to the antibody emits a red-orange fluorescent signal when excited, allowing for the detection and quantification of CD4+ cells in a sample.

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Close spelling variants of this product

Anti-CD4-APC (58 citations) CD4-APC-eFluor 780 (18 citations) Anti-CD4 APC-eFluor 780 (12 citations) CD4 APC-Cy7 (10 citations) Allophycocyanin (APC)-labeled anti-mouse CD4 (6 citations) APC-conjugated anti-mouse CD4 (6 citations) Anti-CD4 APC-Cy7 (6 citations) CD4 APC (clone RPA-T4, (4 citations) Rat anti-mouse CD4 APC (3 citations) CD4 APC‐Alexa Fluor 780 (3 citations)

84 protocols using cd4 apc

Multicolor Flow Cytometry Immunophenotyping

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For titration experiments, cells were stained with anti-human CCR7 IgM (BD Biosciences) and fixable yellow dead cell stain (Invitrogen) at 37 °C for 30 min, washed, and incubated with anti-human CD3 BV650 (Biolegend), CD4 APC (eBioscience), CD45RO ECD (Beckman Coulter), and anti-IgM PE (Invitrogen) at 4 °C for 30 min. For coinfection experiments, cells were stained with anti-human CCR7 IgM (Becton Dickinson) and fixable yellow dead cell stain (Invitrogen) at 37 °C for 30 min, washed, and incubated with anti-human CD3 BV650 (Biolegend), CD4 APC (eBioscience), CD45RA APC-Cy7 (Biolegend), and anti-IgM PE (Invitrogen) at 4 °C for 30 min. Upon completion of antibody incubations, cells were washed with PBS and resuspended in 1 % paraformaldehyde in PBS/BSA prior to analysis using a BD LSRII flow cytometer. At least 50,000 events were collected per sample. FlowJo version 9.6 (Tree Star, Inc.) was used for analysis.

Haqqani A.A., Marek S.L., Kumar J., Davenport M., Wang H, & Tilton J.C. (2015). Central memory CD4+ T cells are preferential targets of double infection by HIV-1. Virology Journal, 12, 184.

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Analyzing T Cell Apoptosis and Activation

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To measure T cell apoptosis, 1 × 10⁶ splenocytes (SPCs) and lymph node cells (LNCs) were prepared and stained with CD4⁺-APC and CD8⁺-PE/CY7 (eBioscience, San Diego, CA, USA) for 30 min at 4°C. After washing with PBS, the cells were stained with Annexin V and propidium iodide according to the manufacturer’s instructions (Dojindo Laboratories, Japan) and analyzed by flow cytometry. To measure T cell activation, the SPCs and LNCs were freshly stained with CD4⁺-APC, CD8⁺-PE/CY7, CD44⁺-PE, and CD69⁺-PE (eBioscience) for 30 min at 4°C and analyzed by flow cytometry. Isotype control staining was also performed as described (21 (link)). Data were analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA).

Ma Y., Yan G., Guo J., Li F., Zheng H., Wang C., Chen Y., Ye Y., Dai H., Qi Z, & Zhuang G. (2021). Berberine Prolongs Mouse Heart Allograft Survival by Activating T Cell Apoptosis via the Mitochondrial Pathway. Frontiers in Immunology, 12, 616074.

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Flow Cytometry Analysis of Immune Cells

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Single-cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi Biotec) for 10 min at 4 °C prior to surface staining. Cell viability was assessed by Fixable viability dye eFluor 520 (eBioscience) to exclude dead cells. The following anti-mouse antibodies were used: FITC-CD11b, PE-F4/80, APC-F4/80, FITC-CD45, PerCP-eFluor 710-MHC Class II (I-A/I-E), PerCP-eFluor 710-CD3, FITC-CD3e, FITC-CD4, APC-CD4, APC-CD8a, PE-PD-L2, PE-B7-H2, PE-B7-H3, PE-B7-H4, PE-TIM-4, PE-VISTA from eBioscience; APC-CD206, PE-PD-L1, APC-CD86, PE/Cy7 Ki-67 from Biolegend; V450-CD4, BV510-CD8, PE-IFN-γ from BD. The following anti-human antibodies were used: PerCP-eFluor 710-CD3, APC-CD4, APC-CD8a from eBioscience; FITC-CD14, PE-PD-L1, APC-CD163, APC-CD86, FITC-HLA-DR, PE-Cy7-CD163, PE-CD25, PE-IFN-γ from Biolegend; FITC-CD8, PE-IL-10 from BD. For intracellular staining, cells were fixed and permeabilized with the Fixation/Permeabilization solution kit (BD). All flow cytometry data was acquired on FACSCalibur or LSRFortessa (BD, San Jose, USA) and analyzed by FlowJo V10 (TreeStar, Ashland, USA).

Wen Z.F., Liu H., Gao R., Zhou M., Ma J., Zhang Y., Zhao J., Chen Y., Zhang T., Huang F., Pan N., Zhang J., Fox B.A., Hu H.M, & Wang L.X. (2018). Tumor cell-released autophagosomes (TRAPs) promote immunosuppression through induction of M2-like macrophages with increased expression of PD-L1. Journal for Immunotherapy of Cancer, 6, 151.

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Multiparameter Flow Cytometry Analysis

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Cell suspensions were treated with anti CD16/CD32 (2.4G2) and then surface stained with combinations of the following fluorochrome-conjugated antibodies: From Biolegend: CD3e–FITC (clone: 145 2C11), Ly6C–FITC or APC/Cy7 (clone: HK1.4), Ly6G–PE/Cy7 (clone: 1A8), CD3e–APC/Cy7 (clone: 145 2C11), CD45R/B220–Pacific Blue (clone: RA3 6B2), CCR2-AlexaFluor647 (clone: SA203G11), CX3CR1-APC (clone: SA011F11), IgG2A,κ-APC isotype control; from BD Biosciences: NK1.1–PerCP/Cy5.5 (clone: PK136); from Ebiosciences: F4/80–APC (clone: BM8), CD4 (L3T4)–FITC (clone: RM4 5), CD11b–FITC (clone: M1/70), CD11b–PE (clone: M1/70), CD4–APC (clone: GK1.5). Cells were acquired on FACSCanto or AriaIII flow cytometers using FACSDiVa software, and data were analyzed using FlowJo software (Tree Star).

Seregin S.S., Golovchenko N., Schaf B., Chen J., Eaton K.A, & Chen G.Y. (2016). NLRP6 function in inflammatory monocytes reduces susceptibility to chemically-induced intestinal injury. Mucosal immunology, 10(2), 434-445.

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Suppressive Capacity of DC-induced T-cells

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To analyze the suppressive capacity of the different DC‐induced T‐cell, cells were harvested from the ASA 96‐well plate on day 7, thoroughly washed and co‐incubated with freshly isolated allogenic responder CD4⁺T‐cell at a ratio of 1:1, 1:2, and 1:4. To track the proliferation of responder T‐cell in the suppression assay, responder cells were pre‐labeled with CFSE (Thermo Fisher Scientific, The Netherlands) according to the manufacturers’ instructions, at a final concentration of 1uM. Dynabeads® human T‐Activator CD3/CD28 (Thermo Fisher Scientific, The Netherlands) were added to activate the responder T‐cell. After 5 days, the mix of ASA cells and CFSE labeled responder CD4⁺ T‐cell was stained with Fixable viability dye eFluor 780 and CD4 APC (both eBioscience, the Netherlands). Suppressive capacity was determined by setting gates on proliferated CD4⁺ T‐cell and comparing the percentage of proliferated cells of different conditions.

Xiao L., van De Worp W.R., Stassen R., van Maastrigt C., Kettelarij N., Stahl B., Blijenberg B., Overbeek S.A., Folkerts G., Garssen J, & van't Land B. (2019). Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells. European Journal of Immunology, 49(7), 1001-1014.

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Evaluating Dexamethasone's Impact on CTL Viability

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Annexin V apoptosis assay was performed to evaluate the effect of dexamethasone on the viability of CTLs from Cas9 control and NR3C1 KO groups. CTLs from both groups were treated with 200 μM dexamethasone (Sigma) for 72 hours. Cells were then collected, washed with Annexin V buffer, and stained with Annexin V (V500; BD Biosciences) and live/dead viability dye (efluor 660; Invitrogen) in addition to CD3 APC Cy7 (Biolegend, Clone HIT3A), CD4 APC (E Biosciences, Clone SK3), and CD8 PerCP Cy5.5 (Biolegend, Clone SK1). The proportion of apoptotic (positive for Annexin V) and dead CTLs (positive for live/dead stain) was determined by flow cytometry.
To confirm the ability of SARS-CoV-2 CTLs to mediate cytotoxicity, autologous PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) per manufacturer’s recommendation and loaded with SARS-CoV-2 Spike (S) (peptide pool 1 or 2), Membrane (M) and Nucleocapsid (N) PepMix (JPT, Germany) [1μg /ml per peptide] overnight. The next day, the SARS-CoV-2 PepMix-loaded PBMCs were co-cultured with the expanded CTLs at 1:1 ratio for 16 hours followed by Annexin V staining as described above. The proportion of dead/apoptotic PBMCs (Live/Dead+ and Annexin V+) was determined by flow cyometry.

Basar R., Uprety N., Ensley E., Daher M., Klein K., Martinez F., Aung F., Shanley M., Hu B., Gokdemir E., Nunez Cortes A.K., Mendt M., Reyes Silva F., Acharya S., Laskowski T., Muniz-Feliciano L., Banerjee P.P., Li Y., Li S., Melo Garcia L., Lin P., Shaim H., Yates S.G., Marin D., Kaur I., Rao S., Mak D., Lin A., Miao Q., Dou J., Chen K., Champlin R.E., Shpall E.J, & Rezvani K. (2021). Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy. Cell Reports, 36(3), 109432.

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Comprehensive Hematopoietic Cell Analysis Protocol

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Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

Gough S.M., Lee F., Yang F., Walker R.L., Zhu Y.J., Pineda M., Onozawa M., Chung Y.J., Bilke S., Wagner E.K., Denu J.M., Ning Y., Xu B., Wang G.G., Meltzer P.S, & Aplan P.D. (2014). NUP98-PHF23 is a chromatin modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD domain histone reader function. Cancer discovery, 4(5), 564-577.

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Multiparametric Flow Cytometry of Murine Splenocytes

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A total of 1 × 10⁶ lymphocytes were acquired and collected from mice spleen, and then 0.5 μg anti-mouse CD16/32 monoclonal antibody (Biolegend, USA) and 5 μL rat serum were used to block the Fc receptor for 10 min. For measuring the number of total CD4⁺ T cell and naïve CD4⁺ T cell, splenocytes were incubated with 1 μg CD3-APC-Cy7 (Biolegend), CD4-APC (eBioscience, Thermo Fisher Scientific, USA), TCRβ-PE and CD62L-FITC (eBioscience, Thermo) for 30 min. For detecting cell activation, cells were stained with 1 μg CD25-PE and CD69-FITC (eBioscience, Thermo). For evaluating cell surface protein expression, EGFR (Thermo)-PE (eBioscience) and Glut1 (Proteintech, China) -FITC (eBioscience) were incubated for 30 min. To measure cell death, splenic cells were stained with PI and Annexin V (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature. Cells were analyzed by FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed by Flowjo10 software (Tree Star, Ashland, OR, USA).

Huang L., Zhang X., Fan J., Liu X., Luo S., Cao D., Liu Y., Xia Z., Zhong H., Chen C., Zhang L., Liu Z, & Tang J. (2022). EGFR promotes the apoptosis of CD4+ T lymphocytes through TBK1/Glut1 induced Warburg effect in sepsis. Journal of Advanced Research, 44, 39-51.

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Multicolor Flow Cytometry Immunophenotyping

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Cultured T-cell lines were stained with fluorochrome-conjugated monoclonal antibodies against CD3 (1 μg), gamma-delta TCR, CD4, CD8, CD19, NK1.1 (0.5 μg each) for subset analysis (all antibodies from BD Bioscience, San Diego, CA). Cells were stained with FOXP3 Alexa488 (1 μg), CD3 PerCP (1 μg) and CD4 APC (0.5 μg) for T-regulatory cells (Treg) analysis according to FOXP3 staining protocol (eBioscience, San Diego, CA). Data acquisition was performed on a FACS Canto flow cytometer (BD Biosciences) and was analyzed using the FlowJo software (Tree Star, OR).

Phan-Lai V., Dang Y., Gad E., Childs J, & Disis M.L. (2015). The anti-tumor efficacy of IL-2/IL-21-cultured polyfunctional neu-specific T-cells is TNF-alpha/IL-17 dependent. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(9), 2207-2216.

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FACS, IHC, and Western Blot Analysis of Mesenteric Lymph Node Cells

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FACS analysis of mesenteric lymph node cells: CD4-APC (eBioscience, clone GK1.5), TCRb-PE (eBioscience, clone H57-597), CD69-biotin (eBioscience, clone H1.2F3), Streptavidin-FITC, (eBioscience). Immunohystochemistry: F4/80 (AbD Serotec; MCA497). Western blot: SIRT2 (H-95, SantaCruz sc-20966), Acetyl-NF-κB (Acetyl-K310, Abcam, ab19870), phospho-IKbα (ser32/36, Cell Signaling 9246), IKbα (L35A5, Cell Signaling 4814), Hsp90 (BD Transduction Laboratories, 610418).

Lo Sasso G., Menzies K.J., Mottis A., Piersigilli A., Perino A., Yamamoto H., Schoonjans K, & Auwerx J. (2014). SIRT2 Deficiency Modulates Macrophage Polarization and Susceptibility to Experimental Colitis. PLoS ONE, 9(7), e103573.

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