Anti human cd4 apc

Manufactured by Thermo Fisher Scientific

Sourced in China

Anti-human CD4-APC is a fluorescent-labeled antibody that binds to the CD4 surface marker on human cells. It is used for the identification and enumeration of CD4+ T cells in flow cytometry applications.

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7 protocols using anti human cd4 apc

Multicolor Flow Cytometry Analysis of PD-1 and PD-L1 Expression

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FITC anti human CD279 (PD-1) (Catlog: 329904, CA), PE Anti Human CD274 (PD-L1, B7-H1) (Catlog: 4307499) fluorescent antibodies were purchased from eBioscience, APC anti human CD4 (Catlog: H10041-11I, Tianjin, China), PE/Cy7 anti-human CD8 (Catalog: H20081-17H) fluorescent antibodies were purchased from Tianjin Sanjian Biological Co., Ltd. Red cell lysate was purchased from Beijing Leigen Biotechnology Co., Ltd., and multi-color flow cytometer was purchased from American Beckman Company. The human peripheral blood lymphocyte isolation solution was purchased from Tianjin Haoyang Biological Co., Ltd. The soluble PD-L1 (sPD-L1) enzyme-linked immunosorbent assay (ELISA) kit was purchased from American Abcam Company, the mouse anti-human PD-1 antibody (primary antibody) was purchased from Beijing Zhongshan Jinqiao Company, the rabbit anti-human PD-L1 [28-8] antibody (primary antibody) (catalog: ab277712, Cambridge, UK) was purchased from American Abcam Company, and the anti rabbit/mouse (secondary antibody) (Envision^™ Detection Kit (catalog: K5007, Copenhagen, Denmark)) universal immunohistochemistry test kit was purchased from Danish DAKO Company.

Feng X., Luo X., Yang Y., Fan Y, & Ye Q. (2023). Expression of PD-1/PD-L1 in peripheral blood and tumor tissues of patients with classical Hodgkin’s lymphoma. Medicine, 102(44), e35757.

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Immunomodulatory Effects of MSCs on T Cell Activation

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PBMCs were isolated from healthy subjects or SLE patients and labelled with 5 μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, eBioscience, Cat# 65–0850-84). Labelled PBMCs were cultured with/without MSCs or AC-MSCs at a ratio of 10:1 in the complete RPMI 1640 medium or cultured in the indicated conditioned medium supplemented with 10% FBS, in the presence of 1 μg/ml anti-CD3/CD28 antibodies (RRID: AB_468854/AB_468926). After 4 days, PBMCs were harvested and stained with APC-anti-human CD4 (eBioscience, RRID: AB_1272048) to detect the CFSE dilution using flow cytometry.

Zhang Z., Huang S., Wu S., Qi J., Li W., Liu S., Cong Y., Chen H., Lu L., Shi S., Wang D., Chen W, & Sun L. (2019). Clearance of apoptotic cells by mesenchymal stem cells contributes to immunosuppression via PGE2. EBioMedicine, 45, 341-350.

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Multiparameter Flow Cytometry Staining

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Cells were washed in FACS medium (PBS containing 2% FBS), and stained with primary antibodies including anti-human CD14-APC (eBioscience, 61D3, 1:100), anti-human CD4-APC (eBioscience, RPA-T4, 1:100), anti-human CD3-FITC (BioLegend, HIT3a, 1:100), anti-human CD8-PE (BD Pharmingen, 555367, 1:100), anti-human CD69-APC (BioLegend, FN50, 1:100), anti-human CD86-PE (BioLegend, IT2.2, 1:100), anti-CD206-APC (eBioscience, 19.2, 1:100), anti-CD11c (Biolegend, Bu15, 1:100), anti-hLAIR1 (eBioscience, NKTA255, 1:100), anti-CD45 (Biolegend, 2D1, 1:100), anti-CD25 (Biolegend, BC96, 1:100), or anti-CD127 (Biolegend, A019D5, 1:100). Flow data were analyzed by Flowjo software. Propidium iodide (PI) staining was used to exclude dead cells in analysis.

Xie J., Gui X., Deng M., Chen H., Chen Y., Liu X., Ku Z., Tan L., Huang R., He Y., Zhang B., Lewis C., Chen K., Xu L., Xu J., Huang T., Liao X.C., Zhang N., An Z, & Zhang C.C. (2022). Blocking LAIR1 signaling in immune cells inhibits tumor development. Frontiers in Immunology, 13, 996026.

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Multiparameter Flow Cytometry Analysis

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Primary antibodies used for this study includes Annexin V-FITC (Biolegend Cat# 640906), anti-PD-1-PE (BioLegend Cat# 621607), anti-human CD8-PE (BioLegend Cat# 344706), anti-mouse CD4-PE (BioLegend Cat# 100408), anti-mouse CD25-PE (BioLegend Cat# 102012), anti-mouse FOXP3-Alexa Fluor® 488 (BioLegend Cat# 126405), anti-mouse CD8-APC (BioLegend Cat# 100711), anti-mouse PD-1-PE (BioLegend Cat# 135205), IgG isotype control-PE (BioLegend Cat# 400907), anti-human CD4-APC (eBioscience Cat# RPA-T4). FoxP3 staining used FoxP3 buffer (BioLegend Cat#421403). Samples were analyzed using FACSCalibur and FlowJo software.

Wu G., Deng W., Chen H.Y., Cho H.J, & Kim J. (2024). Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1. Scientific Reports, 14, 6625.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed on a BD LSRFortessa cytometer, and data were analyzed using FlowJo software. The antibodies used, including anti-human CD3-PE-cyanine 7 (clone: UCHT1), anti-human CD4-APC (clone: GK1.5), anti-human CD4-APCcy7 (clone: GK1.5), anti-human CD8-PE (clone: 53–6.7), anti-human CD8-PEcpcy5.5 (clone: 53–6.7), anti-human CD25-PE (clone: PC61.5), anti-human CD69-APC (clone: H1.2F3), anti-human CD19-APC (clone: 1D3) and anti-human PD-L1-APC (clone: M1H1) were purchased from eBioscience. Anti-human Mesothelin-PE (clone: sc-33,672) was purchased from Santa Cruz Biotechnology. All FACS staining was performed on ice for 30 min, and cells were then washed with PBS containing 1% FBS before cell cytometry. PB, spleen and tumor samples from xenograft mice were treated with a red blood cell lysis buffer (Biolegend), and the cells were stained with the corresponding antibodies.

Qin L., Zhao R., Chen D., Wei X., Wu Q., Long Y., Jiang Z., Li Y., Wu H., Zhang X., Wu Y., Cui S., Wei W., Yao H., Liu Z., Cao S., Yao Y., Zhang Z, & Li P. (2020). Chimeric antigen receptor T cells targeting PD-L1 suppress tumor growth. Biomarker Research, 8, 19.

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CAR T Cell Transduction Protocol

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T cells were enriched and stimulated using human T activator CD3/CD28 Dynabeads (Thermo Fisher) for 2 days in T cell medium with FBS: RPMI, 10% FBS, 1% pen/strep, 1% glutamine, 1% sodium pyruvate, 1% NEAA, 10 mM HEPES, 16.6 μg/ml Gentamycin (all Thermo Fisher), supplemented with 300 U/ml IL2. 0.45 µm-filtered retrovirus cell culture supernatant from stable producer cell lines was centrifuged at 2000xg, 32°C for 2h on non-tissue culture-treated plates (Corning) coated with 20 µg/ml RetroNectin for 2h (Takara). Retrovirus cell culture supernatant was removed, and T cells were spinoculated onto the virus-coated plate at 1000xg for 10’. A 2^nd transduction was performed after 24h. CAR expression was determined by flow cytometry with anti-human CD4-APC (eBioscience), anti-human CD8-PB (BioLegend), and anti-HA-PE (BioLegend), diluted in PBS with 0.1% BSA (Sigma-Aldrich). Cells were analyzed using a CytoFLEX S (Beckman Coulter), and data were analyzed with FlowJo 10.4 software.

Schreiber S., Dressler L.S., Loffredo-Verde E., Asen T., Färber S., Wang W., Groll T., Chakraborty A., Kolbe F., Kreer C., Kosinska A.D., Simon S., Urban S., Klein F., Riddell S.R, & Protzer U. (2024). CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells. Frontiers in Immunology, 15, 1340619.

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Isolation and Expansion of HBV-Specific T Cells

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T cells were restimulated with the respective peptide (1 μM) and stained with the TNF-α and/or IFN-γ secretion assay (Miltenyi Biotec) according to the manufacturer's instructions as well as anti-human CD4-APC (eBioscience, Thermo Fisher Scientific) and anti-human CD8-PB (BioLegend). TNF-α⁺ CD4⁺ T cells were enriched using a fluorescence-activated cell sorting (FACS) Aria III (BD) or a MoFlo XDP cell sorter (Beckmann Coulter), and 0.5 cells/well were seeded in 96-well round-bottom plates containing 7.5 × 10⁴ irradiated heterologous PBMCs (35 Gy), 1 × 10⁴ B-LCLs (50 Gy), 50 IU/mL IL-2, and 30 ng/mL OKT-3 antibody (eBioscience, Thermo Fisher Scientific). HBV-specific T cells clones were identified as described in the supplemental methods. For expansion, selected HBV-specific T cell clones were moved to a 12-well plate containing 5 × 10⁶ irradiated PBMCs, 1 × 10⁶ irradiated B-LCLs, and 30 ng/mL OKT-3 antibody. Then 50 U/mL IL-2 were supplemented on days 1, 5, 8, and 11 and split to two wells when necessary. The TCR chains of HBV-specific clones were analyzed and cloned as described in the supplemental methods.

Schreiber S., Honz M., Mamozai W., Kurktschiev P., Schiemann M., Witter K., Moore E., Zielinski C., Sette A., Protzer U, & Wisskirchen K. (2021). Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors. Molecular Therapy. Methods & Clinical Development, 23, 476-489.

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