Cd3 fitc 17a2

Manufactured by BioLegend

CD3-FITC (17A2) is a fluorescent-labeled antibody that binds to the CD3 antigen. CD3 is a protein complex expressed on the surface of T cells and is involved in the activation and function of these cells. The FITC (fluorescein isothiocyanate) label allows for the detection and analysis of CD3-positive cells using flow cytometry or other fluorescence-based techniques.

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4 protocols using cd3 fitc 17a2

Flow Cytometric Analysis of Muscle Inflammation

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For flow cytometric muscle analysis, mice were euthanized by CO2 asphyxiation 7 days after injury to analyze cellular inflammation in the muscle. Tissue was minced, digested in 1mg/mL collagenase IA for 45 min at 37°C, filtered through membranes with 40μm pore size, and resuspended in 3% FBS for immunostaining. Cell suspensions were immunostained for 30 min on ice followed by fixation in 2% PFA for 10 min and addition of CountBrightTM Absolute Counting Beads. The following antibody panel was used: MerTK-PE (clone 108928; R&D Systems), CCR7-PE/Cy7 (clone 4B12; BioLegend), CD3-FITC (17A2; BioLegend), CD25-PerCP/Cy5.5 (clone PC61; BioLegend), Ly6C-APC (clone HK1.4, BioLegend), Ly6G-APC/Cy7 (clone 1A8; BioLegend), CD11c-BV421 (clone N418; BioLegend), CD11b-BV510 (clone M1/70; BioLegend), CD206-BV605 (clone C068C2; BioLegend), CD64-BV711 (clone X54-5/7.1; BioLegend), and CD4-BV785 (clone GK1.5; BioLegend). Samples were run on a BD FACS Aria IIIu cytometer and data was analyzed using FlowJo software. Cells were immunophenotyped according to the following gating scheme: macrophage, MerTK+CD64+; dendritic cell, NOT(MerTK+CD64+)CD11c+; monocyte, NOT(MerTK+CD64+)CD11c-CD11b+SSClo; neutrophil, Ly6G+SSChi; T lymphocyte, CD3+; helper T lymphocyte, CD3+CD4+; regulatory T lymphocyte (Treg), CD3+CD4+CD25+.

Krieger J.R., Sok M.C., Turner T.C, & Botchwey E.A. (2018). Delivery of Immunomodulatory Microparticles in a Murine Model of Rotator Cuff Tear. MRS advances, 3(24), 1341-1346.

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Splenocyte Phenotyping for T Cell Subsets

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Spleen was collected from the animals in all groups, and splenocytes were phenotyped for T cells (CD3) and their subtypes (CD4 and CD8). Cells were washed with phosphate-buffered saline containing 4% FBS and incubated with FcR-block anti-mouse CD16/CD32 (Clone:93, eBioscience, San Diego, CA) for 15 minutes at 4°C. Following a washing step, cells were incubated in a predetermined optimized concentration of antibodies for 15 minutes at 4°C while shaking. The following antibody clones were used: CD3-FITC (17A2) (Biolegend, San Diego, CA). Cells were analyzed on BD LSR II (BD Biosciences). Flow cytometry data were analyzed using FlowJo_V10 software (Tree Star, Ashland, OR).

Kumari R., Palaniyandi S., Strattan E., Huang T., Kohler K., Jabbour N., Dalland J., Du J., Kesler M.V., Chen Y.H, & Hildebrandt G.C. (2020). TNFAIP8 Deficiency Exacerbates Acute Graft Versus Host Disease in a Murine Model of Allogeneic Hematopoietic Cell Transplantation. Transplantation, 104(3), 500-510.

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Comprehensive Immune Cell Profiling

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For analysis of surface markers, the single‐cell suspensions of PBMCs or TILs were labelled with fluorochrome‐conjugated monoclonal antibodies specific to the relevant targets for 30 min. The primary antibodies include: anti‐human PE/cy7‐CD3 (OKT3, eBioscience), FITC‐CD4 (OKT4, eBioscience), Alexa Fluor 700‐CD8 (HIT8a, BD), Brilliant Violet 421‐TIGIT (MBSA43, eBioscience), APC‐CD96 (NK92.39, eBioscience), PE‐CD226 (c11A8, BioLegend) and FITC‐CD69 (FN50, BioLegend). For the detection of intracellular markers, cells were cultured with PMA/ionomycin/brefeldin A (eBioscience) for 4 h and then were permeabilised using a fixation/permeabilization kit (eBioscience). The primary antibodies include: anti‐human APC/CY7‐IFN‐γ (4S.B3, eBioscience), Alexa Fluor 647‐TNF‐α (MAB11, eBioscience), Pacific Blue‐granzyme B (QA16A02, BioLegend), and PE‐Ki‐67 (Ki‐67, BioLegend). For mouse cell staining, the primary antibodies include: anti‐mouse APC/cy7‐CD45 (30‐F11, BioLegend), FITC‐CD3 (17A2, BioLegend), PE‐CD4 (GK1.5, BioLegend), Alexa Fluor 700‐CD8 (Mp6‐XT22, BioLegend), PE/cy7‐CD69 (H1.2F3, eBioscience), PE‐IFN‐γ (XMG1.2, eBioscience), Brilliant Violet 421‐TNF‐α (Mp6‐XT22, BioLegend), APC‐IL‐2(JES6‐5H4, BioLegend), APC‐NK1.1 (S17016D, BioLegend) and BV421‐Foxp3 (MF‐14, eBioscience).

Luo B., Sun Y., Zhan Q., Luo Y., Chen Y., Fu T., Yang T., Ren L., Xie Z., Situ X., Liu B., Tang K, & Ke Z. (2024). Combining TIGIT blockade with IL‐15 stimulation is a promising immunotherapy strategy for lung adenocarcinoma. Clinical and Translational Medicine, 14(1), e1553.

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Immunological Signaling Pathway Analysis

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Con A was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant mouse IFNγ and U0126 were purchased from BioLegend (San Diego, CA, USA) and Promega (Madison, WI, USA), respectively. Antibodies used for Western blotting and flow cytometry were listed in Table 1.Table 1

Antibodies for Western blotting and flow cytometry.

Western blotting
Bcl2	Cell Signaling Technology (3498)
GAPDH	Cell Signaling Technology (5174)
p44/42 MAPK (ERK1/2)	Cell Signaling Technology (4695)
phospho-p44/42 MAPK (pERK1/2)(Thr 202/Tyr 204)	Cell Signaling Technology (4370)
STAT1	Cell Signaling Technology (9172)
phospho-STAT1 (Tyr 701)	Cell Signaling Technology (9167)
STAT3	Cell Signaling Technology (12640)
phospho-STAT3 (Tyr 705)	Cell Signaling Technology (9145)
HRP-goat anti-rabbit IgG	Cell Signaling Technology (7074)
HRP-anti-mouse IgG	Cell Signaling Technology (7076)

Flowcytometry

CD16/32	BioLegend (101302)
FITC-CD3 (17A2)	BioLegend (100204)
PE-CD4 (GK1.5)	BD Pharmingen (553730)
FITC-CD8 (53–6.7)	BioLegend (100706)
PE-CD8 (53–6.7)	BioLegend (100708)
PE-NK1.1 (PK136)	BioLegend (108708)
FITC-IFNγ (XMG1.2)	BioLegend (505806)

Sun C., Fujisawa M., Ohara T., Liu Q., Cao C., Yang X., Yoshimura T., Kunkel S.L, & Matsukawa A. (2021). Spred2 controls the severity of Concanavalin A-induced liver damage by limiting interferon-gamma production by CD4+ and CD8+ T cells. Journal of Advanced Research, 35, 71-86.

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