Igg2a fitc

Manufactured by Thermo Fisher Scientific

Sourced in United States

IgG2a-FITC is a fluorescently labeled antibody that can be used to detect and quantify the presence of the IgG2a immunoglobulin subclass. It is used in various immunological assays and flow cytometry applications.

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IgG2a (72 citations) Rat IgG2a K Isotype Control FITC (4 citations) FITC rat IgG2a (3 citations) FITC-anti-mouse-IgG2a (2 citations) FITC mouse IgG2A isotype control (2 citations) Rat IgG2a(κ)-FITC (2 citations)

4 protocols using igg2a fitc

Multi-Color Flow Cytometry Immunophenotyping

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Antibody combination panels for four or five color immune-staining were as follows:

CD3PerCP, CD4 FITC, CD25APC, and rat Foxp3PE (all from eBioscience, San Diego, CA).

CD4 PercpCy5.5, HLA-DR APC, CD127 Pac Blue and rat Foxp3 PE (eBioscience, San Diego, CA).

CD4 PercpCy5.5, CCR5 FITC, CXCR4 APC (Biolegend, San Diego, CA) and Foxp3 PE.

CD4 PercpCy5.5, HIV-1 p24 FITC (Beckman Coulter, Brea, CA), Bcl-2 PE (BD Biosciences, San Jose, CA) and Foxp3 e-fluor660 (eBioscience).

CD4 PercpCy5.5, CD127 Pac Blue, Foxp3 PE, HIV-1 p24 FITC and IFN-γ Alexa Fluor 647 (Biolegend).

Isotype antibodies used included: IgG2a APC, IgG1 Pac Blue, rat IgG2a PE, IgG2a FITC, rat IgG2a AP, IgG1 FITC, IgG1 PE, IgG2a e-fluor660, and IgG1 Alexa Fluor 647.
The protocol and buffer set from eBioscience were used for all experiments involving intracellular staining. All samples were fixed and acquired within 1h of completion of staining, and analysis was by Miltenyi MACSQuant Flow cytometer.
Data were analyzed by FlowJo software (Treestar Inc, Ashland, OR) at the completion of study.

Hirsch C.S., Baseke J., Kafuluma J.L., Nserko M., Mayanja-Kizza H, & Toossi Z. (2016). Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection. Journal of clinical & experimental immunology, 1(1), http://www.opastonline.com/wp-content/uploads/2016/11/expansion-and-productive-hiv-1-infection-of-foxp3-positive-cd4-t-cells-at-pleural-sites-of-hivtb-co-infection-jcei-16-007.pdf.

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Flow Cytometric Analysis of Immune Cell Populations

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Cells isolated from the BAL and other tissues were analyzed via flow cytometry using the following monoclonal antibodies: anti-CD8a-FITC (53-6.7), anti-CD25-PE (PC61), anti-CD4-PeCy7 (RM4-5), anti-CD3e-PerCP-Cy5.5. (145-2C11), and anti-CD19-PerCP-Cy5.5 (1D3) (BD PharMingen, San Diego, CA, USA). Samples were washed in PBS containing 0.2% bovine serum albumin and 0.1% NaN₃. Aliquots containing 10⁴–10⁶ cells were incubated with 100 μl of appropriately diluted antibodies for 30 min at 4°C. After staining, the cells were washed with the above PBS solution, and relative fluorescence intensities were determined on a four-decade log scale by flow cytometric analysis using a LSR II (Becton-Dickinson, San Diego, CA, USA). For the identification of T_reg, intracellular staining of Foxp3 protein was used. Briefly, cells stained with the antibodies mentioned previously (i.e., anti-CD3e, anti-CD4, and anti-CD25) were permeabilized using fixation/permeabilization buffer following the manufacturer’s protocol, and stained using either anti-Foxp3-FITC (FJK-16s) or anti-Foxp3-APC (FJK16s) with corresponding isotype controls, IgG2a-FITC or -APC (eBioscience, San Diego, CA, USA).

Carson WF I.V., Guernsey L.A., Singh A., Secor ER J.r., Wohlfert E.A., Clark R.B., Schramm C.M., Kunkel S.L, & Thrall R.S. (2015). Cbl-b Deficiency in Mice Results in Exacerbation of Acute and Chronic Stages of Allergic Asthma. Frontiers in Immunology, 6, 592.

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Multicolor Immunofluorescence Staining of Lymphoid Tissues

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4 × 10⁶ Tg4 cells were transferred to mice 1 day after PBS/PIT. Spleens were isolated 48 hr later and embedded and frozen in OCT (Cellpath, Newtown, UK). 6 µm thick sections were fixed in ice-cold acetone. Non-specific binding and endogenous biotin were blocked with 3% BSA (Sigma) and an avidin-biotin blocking kit (Vector Laboratories, Peterborough, UK) respectively. Tissue sections were stained with hamster anti-CD11c-biotin (Biolegend, San Diego, CA, USA), rat anti-CD4-FITC and mouse anti-CD45.1-APC (eBioscience). Biotinylated antibodies were detected with streptavidin-Alexa Fluor 555 (Invitrogen). Sections were mounted in Permafluor mounting medium (Thermo Scientific, Hemel Hempstead, UK). To determine specific binding, secondary antibodies alone or isotype controls (IgG-Biotin [Biolegend], IgG2a-FITC, IgG2a-APC [eBioscience]) were used. Images were acquired with a Leica confocal laser scanning microscope SP5 and LAS AF software (Leica, Wetzlar, Germany) and processed with ImageJ/Fiji software (NIH, Bethesda, MD, USA).

McPherson R.C., Konkel J.E., Prendergast C.T., Thomson J.P., Ottaviano R., Leech M.D., Kay O., Zandee S.E., Sweenie C.H., Wraith D.C., Meehan R.R., Drake A.J, & Anderton S.M. (2014). Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerized by peptide immunotherapy. eLife, 3, e03416.

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Multicolor Flow Cytometry for PBMC Analysis

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All blood samples used for this study were collected at the time of admission to the department of general surgery. PBMCs were freshly isolated from 4 ml of heparinized blood using Ficoll density gradient centrifugation. Lymphocytes were re-suspended in PBS supplemented with 2% bovine serum albumin at a concentration of 1 × 10⁶ cells/ml. Cell surface marker analysis was performed using four or five color flow cytometric analysis. Fluorochrome-labeled mouse anti-human monoclonal antibodies against CD3-PC7, CD4-PerCP, and CD25-APC were purchased from Beckman Coulter. Foxp3-Ax488 and IgG2a-FITC (eBioscience, CA, USA) antibodies were used in combination with appropriate isotype controls to identify positive and negative cell populations. For double staining of IL-17 and Foxp3, PBMCs were isolated using Ficoll density gradient separation and stimulated with PMA (50 ng/ml) and ionomycin (1 mg/ml) in the presence of Golgi-Stop reagent for 4–6 h. First, extracellular labels were used, including specific Abs against human CD3, CD4, and CD25. The cells were then fixed and permeabilized with Perm/Fix solution and stained using anti-IL-17A (intracellular) and anti-Foxp3 (intranuclear) antibodies or control isotype antibodies for 30 min on ice. Multicolor FACS analysis was performed using a BD FACS Aria flow cytometer (BD).

Ma Y., Chen L., Xie G., Zhou Y., Yue C., Yuan X., Zheng Y., Wang W., Deng L, & Shen L. (2016). Elevated level of Interleukin-35 in colorectal cancer induces conversion of T cells into iTr35 by activating STAT1/STAT3. Oncotarget, 7(45), 73003-73015.

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