Rat igg2a isotype control

Manufactured by BioLegend

Sourced in United States, Japan

The Rat IgG2a isotype control is a laboratory reagent used as a reference or control sample in immunoassays and other experimental procedures involving rat immunoglobulin G2a (IgG2a) antibodies. It serves as a negative control to help distinguish specific target binding from non-specific background signals.

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Isotype control (56 citations) Rat IgG2a (27 citations) IgG2a (24 citations) IgG2a isotype control (8 citations)

15 protocols using rat igg2a isotype control

Inhibition and Stimulation of Immune Receptors

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All antibodies were administered by a single i.d. injection into the ear pinna using a 30-gauge needle under inhalation anesthesia. For inhibition of Skint1 for confocal imaging, 2 μg of anti-Skint1 (clone 2G2) or rat IgG2a isotype control (BioLegend) were administered in 25 μl of PBS. For all other inhibition experiments, mice were administered 10 μg of anti-Skint1 (BioLegend), anti-4-1BBL (BioXCell), anti-GITRL (BioLegend) or rat IgG2a isotype control (BioLegend) in 10 μl of PBS. For antibody-mediated stimulation of receptors, mice were administered 2 μg of agonistic anti-4-1BB (R&D Systems) and anti-GITR (BioXCell) or respective isotype controls rat IgG2a (BioLegend) and rat IgG2b (BioXCell) in 10 μl of PBS. Anti-Skint1 hybridoma supernatant was produced by the Monoclonal Antibody Core Facility at the Helmholtz Centre Munich, and the antibody was purified by Cell Services at The Francis Crick Institute.

McKenzie D.R., Hart R., Bah N., Ushakov D.S., Muñoz-Ruiz M., Feederle R, & Hayday A.C. (2022). Normality sensing licenses local T cells for innate-like tissue surveillance. Nature Immunology, 23(3), 411-422.

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Feline PBMC Isolation and Functional Analysis

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PBMCs from specific pathogen-free cats (one 3 year olds, four 4 year old and two 6 year old), which are kept in our veterinary teaching hospital or NIPPON ZENYAKU KOGYO Co., Ltd. as a blood donor, were separated by density gradient centrifugation using Lymphoprep (Axis-shield, Oslo, Norway). For PD-1 and PD-L1 expression analysis, the isolated PBMCs were cultured in R10 for 1.5 h to attach monocytes onto the culture dish, and only the lymphocytes in the supernatant were collected. The collected PBLs were cultured in R10 medium in the absence (unstimulated control) or presence of 10 µg/mL of Con A at 37 °C for 48 h. The cells were stained with antibodies for flow cytometric analysis.
For the IFN-γ production assay, PBLs are collected by incubating PBMCs and attaching monocytes as in the flow cytometry analysis, 2 × 10⁵ PBLs of were cultured in R10 medium in the presence of 10 µg/mL of Con A with 10 µg/mL of mouse IgG₁ isotype control (Biolegend) or anti-PD-1 antibody (1A1-2), or rat IgG_2a isotype control (Biolegend) or anti-PD-L1 antibody (G11-6), or cat IgG isotype control (Jackson ImmunoResearch Laboratories) or anti-PD-1 chimeric antibody (ch-1A1-2) at 37 °C for 48 h. The cell supernatants were collected, and the amount of IFN-γ was measured with DuoSet ELISA Development System feline IFN-γ (R&D systems, Inc.).

Nishibori S., Kaneko M.K., Nakagawa T., Nishigaki K., Kato Y., Igase M, & Mizuno T. (2023). Development of anti-feline PD-1 antibody and its functional analysis. Scientific Reports, 13, 6420.

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Multiparametric Flow Cytometry Analysis of Mediastinal Lymph Nodes

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Mediastinal lymph nodes (mLNs) were harvested, and single-cell suspensions were prepared. The cells were preincubated with Fc blocker and then incubated on ice for 30 min with antibody cocktail and Fixable Viability Dye, Ghost Red 780 (Tonbo Biosciences). Flow cytometry was conducted using fluorochrome-conjugated Abs for CD3 (145-2C-11, BD Biosciences), CD4 (RM5, BD Biosciences), CXCR5 (2G8, BD Biosciences), ICOS (C398.4A, BioLegend), PD-1 (J43, BD Biosciences and 29F.1A12, BioLegend), B220 (RA3–6B2, BD Biosciences), Fas (Jo2, BD Biosciences), PNA (FL-1071, Vector Laboratories), CD11b (M1/70, BD Biosciences), Ly6G (1A8, BioLegend), and rat IgG2a isotype control (BioLegend). Data were acquired using Fortessa (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). All data were analyzed with the FlowJo software (TreeStar, Ashland, OR).

Lama J.K., Iijima K., Kobayashi T, & Kita H. (2022). Blocking the inhibitory receptor PD-1 prevents allergic immune response and anaphylaxis in mice. The Journal of allergy and clinical immunology, 150(1), 178-191.e9.

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Flow Cytometric Analysis of Integrin Expression

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Integrin surface expression was determined by FACS using a LSRFortessa^TM X-20 flow cytometer (BD Biosciences). Staining and measurement were performed in PBS supplemented with 2% FBS and 2 mM EDTA. Cells were incubated with biotinylated anti-integrin antibodies and subsequently with Cy5-labeled strepdavidin (Catalog# 016-170-084, Jackson ImmunoResearch Laboratories). Data were analyzed using FlowJo software. The following antibodies were used for flow cytometry: hamster IgM anti-integrin β1 (Catalog# 13-0291-82, eBioscience), rat IgG2a anti-integrin α6 (Catalog# 13-0495-82, eBioscience), rat IgG1 isotype control (Thermo Fisher Scientific), hamster IgG isotype control (Thermo Fisher Scientific), hamster IgG anti-integrin β3 (Catalog# 553345, BD Pharmingen), rat IgG2a anti-integrin α5 (Catalog# 557446, BD Pharmingen^TM), rat IgG1 anti-integrin αV (Catalog #104104, BioLegend), hamster IgM isotype control (BioLegend), and rat IgG2a isotype control (BioLegend). All listed antibodies were biotinylated and used at 1:200 dilution.

Zhu L., Yang J., Bromberger T., Holly A., Lu F., Liu H., Sun K., Klapproth S., Hirbawi J., Byzova T.V., Plow E.F., Moser M, & Qin J. (2017). Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation. Nature Communications, 8, 1744.

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Blocking rBM Interactions on Cell Clustering

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To determine the effects of blocking cellular interactions with specific rBM components 12Z were trypsinized, resuspended, and incubated with 10 μg/mL anti-α6 integrin function blocking antibody (GoH3, Biolegend, San Diego, CA), 50 μg/mL soluble collagen IV, 30 mM melibiose (D(+)-Melibiose monohydrate, Acros Organics, Fair Lawn, NJ), or a rat IgG2a isotype control (Biolegend) for 45 minutes at room temperature. Cells were then plated in rBM as described above with the exception that each overlay was modified to include the blocking treatments and imaged at 24 hours. To quantify the effect of each inhibitor, images were taken in the middle of each well (n=3) and clusters were binned as either small clusters (2–5 cells/cluster) or large clusters (>6 cells/cluster).

Pollock K., Jaraczewski T.J., Carroll M.J., Lebovic D.I, & Kreeger P.K. (2014). Endometriotic Epithelial Cell Response to Macrophage-Secreted Factors is Dependent on Extracellular Matrix Context. Cellular and molecular bioengineering, 7(3), 409-420.

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Obesity Induction and CD169+ ATM Depletion in Mice

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C57BL/6J, CCR2^−/−, and CX₃CR‐1^GFP (B6.129P2(Cg)‐Cx3cr1^tm1Litt/J) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6 CD169‐DTR mice were generated in our laboratory (Purnama et al, 2014 (link)) and crossed with CCR2^−/− to obtain the final CCR2^−/−CD169‐DTR mouse line. We used male mice only for these experiments. To induce obesity, mice aged 5–6 weeks were given a high‐fat diet (HFD) that comprised 60% kcal of fat (Research Diet, D12492, New Brunswick, NJ, USA) for 8, 12, or 16 weeks; age‐matched control mice were given a normal chow diet (ND).
CD169‐DTR⁺ and CD169‐DTR⁻ (WT) mice were intraperitoneally (i.p.) injected with 20 ng/g DT (Sigma) every 3–4 days to maintain the depletion of CD169⁺ ATMs. C57BL/6J mice were i.p. injected with rat IgG2a isotype control (BioLegend) or anti‐CSF1R blocking antibody (Clone AFS98, BioXCell, West Lebanon, NH, USA), twice per week (400 μg per mouse) for 12 days. No randomization nor blinding was used during our experiments.
All mice were bred and maintained in a specific‐pathogen‐free animal facility at Nanyang Technological University (NTU), Singapore. The mice experiments were carried out in strict accordance with the recommendations of the NACLAR (National Advisory Committee for Laboratory Animal Research) and approved by the Institutional Animal Care and Use Committee (IACUC) of NTU (ARF‐ SBS/NIE A18014).

Chen Q., Lai S.M., Xu S., Tan Y., Leong K., Liu D., Tan J.C., Naik R.R., Barron A.M., Adav S.S., Chen J., Chong S.Z., Ng L.G, & Ruedl C. (2021). Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice. EMBO Reports, 22(8), e52835.

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Anti-PD-1 Immunotherapy for Parasitic Infection

Cited 1 time

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To block PD-1 in vivo, 100 μg of rat anti-mouse PD-1 mAb (clone 29F.1A12; Biolegend, San Diego, CA), rat IgG2a isotype control (Biolegend) or PBS was injected intraperitoneally (i.p.) every three days, starting 24 d post-infection until 3 d before the mice were sacrificed [26 (link)]. At 42 d post-infection, all the mice were sacrificed. Serum samples were collected for ELISA detection of IL-4 levels, and splenocytes were prepared for FCM and qPCR analysis. In addition, livers were isolated for a pathological examination and an assessment of their egg burden.

Zhou S., Jin X., Li Y., Li W., Chen X., Xu L., Zhu J., Xu Z., Zhang Y., Liu F, & Su C. (2016). Blockade of PD-1 Signaling Enhances Th2 Cell Responses and Aggravates Liver Immunopathology in Mice with Schistosomiasis japonica. PLoS Neglected Tropical Diseases, 10(10), e0005094.

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Multiparameter Flow Cytometry Analysis

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For surface staining, cell suspensions were incubated with
anti-CD16/CD32 (2.4G2) followed by staining with antibodies against CD45.2,
CD31, Ter119, MHC class II (I-A/I-E), CD69, CD326 (EpCAM), CD19, CD3,
TCRβ TCRγδ, CD4, CD8, NKp46, Ly6G, CD11c, CD64, CD11b,
CD40, CD80, CD86, Rat IgG2a isotype control, Armenian Hamster IgG isotype
control, Rat IgG2b isotype control and T-bet (all BioLegend); CD90.1, YAe and
CD200r (eBioscience); Vβ6, CD19, DX5 (CD49b), NK1.1, Ly6C and
IFNγR1 (CD119) (BD Biosciences). 7AAD (Sigma) was added before cell
acquisition. For intracytoplasmic staining (villin and vimentin (Abcam,
Cambridge, UK) and αSMA (eBioscience)), single-cell suspensions were
incubated with fixable live/dead cell dye (ThermoFisher), FcγR blocked
with 2.4G2 and stained for surface markers. Cells were then fixed and
permeabilized using the BD Fix/Perm kit (BD) according to the
manufacturer's protocol and stained with Abs against villin, vimentin or
αSMA for 30 min at room temperature, washed and acquired. T-bet
expression was determined using the Foxp3 staining buffer set (eBioscience)
according to the manufacturer's protocol. Samples were acquired with a BD
LSRFortessa (BD) and analysis was performed with FlowJo v9 (Tree Star) software.
Representative flow cytometry plots display concatenated replicates from one
experiment.

Koyama M., Mukhopadhyay P., Schuster I.S., Henden A.S., Hülsdünker J., Varelias A., Vetizou M., Kuns R.D., Robb R.J., Zhang P., Blazar B.R., Thomas R., Begun J., Waddell N., Trinchieri G., Zeiser R., Clouston A.D., Degli-Esposti M.A, & Hill G.R. (2019). MHC class II antigen presentation by the intestinal epithelium initiates graft-versus-host disease and is influenced by the microbiota. Immunity, 51(5), 885-898.e7.

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Delayed-Type Hypersensitivity Assay in Mice

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Mice were treated by intraperitoneal injection of 100 µg of rat-anti-mouse IL-12/23p40 antibody (clone: C17.8, Biolegend) or of 100 µg of rat IgG2a isotype control (Biolegend) once. One day later a DTH response to OVA was induced by sensitizing mice as described in the previous section and in Figure 7A. Ten days later mice were anesthetized with isoflurane and the ear thickness of the untouched, left ear was measured using a caliper. Subsequently, mice were challenged by injection of 50 µg of OVA in the left ear skin. 48 hours after the challenge the ear thickness was again measured, and mice were sacrificed for further analysis.

Aebischer D., Willrodt A.H, & Halin C. (2014). Oxazolone-Induced Contact Hypersensitivity Reduces Lymphatic Drainage but Enhances the Induction of Adaptive Immunity. PLoS ONE, 9(6), e99297.

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Phenotyping Immune Cells in Peripheral Blood and Lymph Nodes

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Peripheral blood mononuclear cells (PMBCs) were obtained from blood and suspended in 100 μL FACS buffer containing DPBS and 2% FBS. The PBMCs and lymphocytes derived from dLNs were blocked using an FcR-blocking Reagent (BioLegend, San Diego, CA, USA) and divided into two groups. One group was stained with a surface marker panel containing CD3, CD4, CD25, CD8, CD44, CD45 (BioLegend), and intracellular markers Granzyme-B and FoxP3 (BioLegend). The other group was stained for F4/80, MHC-II, CD86, CD11b, CD11c, NK1.1, Ly6-G, and CD45 (BioLegend). Rat IgG2b isotype control (BioLegend) was used for CD3, CD11b, CD4, CD11c, CD25, Gr-1, CD44, CD45, FoxP3, and Granzyme B; rat IgG2a isotype control (BioLegend) for CD86 and F4/80, CD8a and NK1.1; and rat IgG1 isotype control (BioLegend) for MHC II. Staining with intracellular markers required fixation and permeabilization of cells following extracellular staining. Samples were analyzed on a Beckman Flow Cytometer in the Beckmann FACS facility.

Jin M., Huo D., Sun J., Hu J., Liu S., Zhan M., Zhang B.Z, & Huang J.D. (2024). Enhancing immune responses of ESC-based TAA cancer vaccines with a novel OMV delivery system. Journal of Nanobiotechnology, 22, 15.

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