Rat igg2a isotype control 2a3

Manufactured by BioXCell

Sourced in United States

The Rat IgG2a isotype control (2A3) is a laboratory reagent used as a control in immunological experiments involving rat antibodies. It serves as a reference point to distinguish specific antibody binding from non-specific background signals. The core function of this product is to provide a standardized control for the evaluation and interpretation of experimental results.

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15 protocols using rat igg2a isotype control 2a3

Targeting Notch1 and Dll4 in Tumor Angiogenesis

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Research-grade, function-blocking antibodies to mouse Notch1 and Dll4 (Kuhnert et al., 2015 (link)) were generated using VelociImmune^® technology. Anti-Notch1 (10 mg/kg body weight, twice a week), Dll4 (5 mg/kg body weight, once a week) and control antibodies (10 mg/kg body weight, twice a week) were administered intraperitoneally (IP). Anti-PD-1 (RMP1-14) and isotype control rat IgG2a (2A3) were purchased from BioXcell and administered IP at 10 mg/kg body weight twice a week starting at 3 weeks post-transplantation. For immunofluorescent staining, anti-CD31 (ab28364, Abcam), anti-N1-ICD (D3B8, Cell Signaling Technology) and anti-Dll4 (AF1389, R&D Systems) were used.

Gao J., Van Meter M., Hernandez Lopez S., Chen G., Huang Y., Ren S., Zhao Q., Rojas J., Gurer C., Thurston G, & Kuhnert F. (2019). Therapeutic targeting of Notch signaling and immune checkpoint blockade in a spontaneous, genetically heterogeneous mouse model of T-cell acute lymphoblastic leukemia. Disease Models & Mechanisms, 12(9), dmm040931.

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Mouse Immune Response Evaluation

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Purified monoclonal rat-anti-mouse IL-7Rα (A7R34) and its isotype control rat-IgG2a (2A3) used for injection were obtained from BioXCell. For flow cytometry, fluorescence conjugated anti-CD4, anti-CD8, anti-CD19, anti-IFN-γ, anti-PD-1, anti-IL-17 and anti-CD16/32 antibodies were purchased from BioLegend, and fluorescence conjugated anti-Foxp3 antibody was obtained from eBioscience. For Immunohistochemical staining, biotin-conjugated anti-CD4 and anti-CD8 antibodies were obtained from eBiosience and biotin-conjugated anti-B220 antibody was obtained from BioLegend; anti-TNF-α, anti-claudin-1 and anti-claudin-2 antibodies were purchased from Abcam. For immunofluorescence staining, anti-aquaporin-5 (AQP5) antibody and Alexa Fluor647-conjugated anti-rabbit IgG were purchased from Abcam.

Zhou J, & Yu Q. (2018). Anti-IL-7 receptor-α treatment ameliorates newly established Sjögren’s-like exocrinopathy in non-obese diabetic mice. Biochimica et biophysica acta, 1864(7), 2438-2447.

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Tumor Induction and Immunotherapy Protocol

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All mice were anesthetized with subcutaneous administration of ketamine and xylazine. After skin incision, mice were then inoculated in the left lung with 1 × 10⁶ KP^OVA cells in 20 μL PBS mixed with 20 μL Matrigel (Corning). For ABx treatment, mice were treated with a cocktail of ampicillin (1 g/L), neomycin (1 g/L), vancomycin (0.5 g/L), and metronidazole (0.5 g/L), or with water sterilized by autoclave, starting 2 weeks before tumor injection and continuing throughout the experiment. Mice undergoing anti–PD-1 therapy were i.p. injected with anti-mouse PD-1 antibody (RMP1-14; BioXcell) for 2 weeks (3 times/week) starting 3 days after tumor injection. Control groups were injected with rat IgG2a isotype control (2A3; BioXcell) in the same manner.

Masuhiro K., Tamiya M., Fujimoto K., Koyama S., Naito Y., Osa A., Hirai T., Suzuki H., Okamoto N., Shiroyama T., Nishino K., Adachi Y., Nii T., Kinugasa-Katayama Y., Kajihara A., Morita T., Imoto S., Uematsu S., Irie T., Okuzaki D., Aoshi T., Takeda Y., Kumagai T., Hirashima T, & Kumanogoh A. (2022). Bronchoalveolar lavage fluid reveals factors contributing to the efficacy of PD-1 blockade in lung cancer. JCI Insight, 7(9), e157915.

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Tumor Growth Inhibition by Anti-CD8α Therapy

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NC mice with tumors within a uniform size range (150–300 mm³) were injected with 200 μg of anti-CD8α (53–6.72, Bio X Cell) or rat IgG2a isotype control (2A3, Bio X Cell) starting on the day that each tumor was first identified (150–300mm³), and continuing twice per week until the study endpoint. 24 hours after antibody delivery, mice were administered either CP-Dox or PBS control, as described above. Tumor growth was determined by caliper measurement 3 times weekly. For flow cytometry analysis, tumor tissue was stained with antibodies against CD45 (BV605, 30F11), CD11b (PE, M1/70), CD11c (BV421, N418), CD3e (PE-Cy7, 145–2C11), CD4 (AF700, GK1.5), and CD8 (PerCP Cy5.5, 53–6.7) and analyzed by flow cytometry. All flow cytometry reagents were purchased from Biolegend.

Dodd R.D., Scherer A., Huang W., McGivney G.R., Gutierrez W.R., Laverty E., Ashcraft K.A., Stephens V.R., Yousefpour P., Saha S., Knepper-Adrian V., Floyd W., Chen M., Ma Y., Mastria E.M., Cardona D.M., Eward W.C., Chilkoti A, & Kirsch D.G. (2020). Tumor subtype determines therapeutic response to chimeric polypeptide nanoparticle-based chemotherapy in Pten-deleted mouse models of sarcoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(18), 5036-5047.

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Modulation of Immune Response via Cytokine Neutralization

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Anti-IL-2 Ab (clones S4B6 and JES6-1A12) and rat IgG2a isotype control (2A3) were purchased from Bio X Cell. Anti-IL-15 (M96) was a kind gift from Amgen. For IL-2 and IL-15 neutralization, 3 week old mice were injected i.p with 150 μg of S4B6 and Jes61A12 and 25 μg of M96 or with 150 μg isotype control (2A3), on days 0, 2, 4 and 6.The mice were sacrificed on day 7 and thymii and spleen were harvested.

Shanmuganad S., Hummel S.A., Varghese V, & Hildeman D.A. (2022). Bcl-2 is necessary to counteract Bim and promote survival of TCRαβ+ CD8αα+ IEL precursors in the thymus. Journal of immunology (Baltimore, Md. : 1950), 208(3), 651-659.

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Immunohistochemical Staining of Cryosections

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Cryosections 7um in thickness were cut and fixed in cold acetone.
Sections were stained with anit-ICAM1 (3E2; BD Biosciences); anti-VCAM1 (429; BD
Biosciences); anti-MADCAM1(MECA367; Bio X Cell); anti-IgD (11-26c.2a;
Biolegend); anti-Vitronectin (347317; R&D); rat IgG2a isotype control
(2A3; Bio X Cell) or anti-CD35 (8C12; BD Biosciences) using described protocols
(36 (link)).

Wang X., Rodda L., Bannard O, & Cyster J.G. (2014). Integrin-mediated interactions between B cells and follicular dendritic cells influence germinal center B cell fitness. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4601-4609.

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Multiparameter Flow Cytometry Analysis of Germinal Center B Cells

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Spleen and lymph nodes were isolated and mashed into media containing
2% FCS. For analysis of GC B cells, cells were stained with anti-B220
(RA3-6B2; BD Biosciences or Biolegend); anti-CD19 (6D5; BD Biosciences);
anti-Fas (Jo2; BD Biosciences); anti-IgD (11-26c.2a; BD Biosciences or
Biolegend); anti-CD45.1 (A20; BD Biosciences or Biolegend); anti-CD45.2 (104; BD
Biosciences or Biolegend); anti-IgG2b (RMG2b-1; BD Biosciences); homemade
Alexa647 conjugated DEL; antibody to T cell and B cell activation antigen (GL7;
BD Biosciences); anti-Mouse Eα52-68 peptide bound to I-A^b(Y-Ae, eBioscience); anti-integrin β1 (MB1.2; Chemicon); anti-integrin
β2 (C71/16; BD Biosciences); anti-integrin β3 (2C9.G2;
Biolegend); anti-integrin β7 (M293; BD Biosciences); anti-integrin
α4 (PS/2; Bio X Cell); anti-integrin α_L (M17/4; Bio X
Cell); anti-integrin α_V (RMV-7; BD Biosciences); rat IgG2a
isotype control (2A3; Bio X Cell); rat IgG2b isotype control (LTF-2; Bio X Cell)
or rat IgG1 isotype control (R3-34; BD Biosciences). BrdU staining was done
using BrdU flow kit (BD Biosciences) following manufacturer's
instructions. For intracellular staining of phosphorylated AKT at Ser473 (pAKT),
cells were instantly fixed and stained as described (35 (link)). Anti-pAKT (9271; Cell Signaling Technology) was
used.

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Orthotopic Tumor Implantation and Therapeutic Evaluation

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Tumor cells were implanted orthotopically as previously described (15 (link)), using 6- to 8-week-old C57BL/6J mice (Charles River Laboratories) or B6.129S7-Rag1^tm1Mom/J mice (The Jackson Laboratory). Mice were monitored daily for weight loss or symptom appearance and euthanized according to veterinarian-authorized endpoints. For bioluminescence monitoring, mice were injected intraperitoneally (IP) with 3 mg of Luciferin (Gold Biotechnology) 5 minutes prior to image acquisition on an IVIS Spectrum system (Xenogen, PerkinElmer) with 3 images collected at 5-minute intervals. Only the highest signal was used for quantification. Therapeutic Ab were all purchased from Bio X Cell: anti–PD-1 (RMP1-14), anti–CTLA-4 (9D9), anti-CD40 (FGK4.5), rat IgG2a isotype control (2A3), and mouse IgG2b isotype control (MPC-11) were injected IP as specified. The prodrug BAL101553 was provided by Basilea Pharmaceutica International Ltd., administered by oral gavage at the indicated concentrations. TMZ was purchased from MilliporeSigma and injected IP at 50 mg/kg dose.

Genoud V., Espinoza F.I., Marinari E., Rochemont V., Dietrich P.Y., McSheehy P., Bachmann F., Lane H.A, & Walker P.R. (2021). Treating ICB-resistant glioma with anti-CD40 and mitotic spindle checkpoint controller BAL101553 (lisavanbulin). JCI Insight, 6(18), e142980.

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Multi-Marker Immune Cell Phenotyping

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Pacific Blue–conjugated anti‐human CD4 (RPA‐T4) and FITC‐ and Pacific Blue–conjugated anti‐mouse CD4 (GK1.5) were purchased from BioLegend. PE‐conjugated, APC‐conjugated, or Pacific Blue–conjugated anti‐mouse Foxp3 antibody (FJK‐16S), FITC‐conjugated anti‐human TCR (IP26), PE‐conjugated anti‐human CD25 (BC96), PerCP‐Cyanine5.5–conjugated anti‐human CD4 (OKT4), eFluoro 660–conjugated anti‐human Foxp3 (PCH101), PE‐conjugated anti‐mouse PD‐1 (J43), and anti‐IFN‐γ (R4‐6A2) were purchased from eBioscience. APC‐conjugated anti‐human CD8a (RPA‐T8) and PE‐conjugated anti‐mouse RORγt (B2D) were purchased from Invitrogen. p‐S6 (S235/236), ribosomal protein (D57.2.2E), S6 ribosomal protein (54D2), and GAPDH (D16H11) were obtained from Cell Signaling Technology. Anti‐mouse PD‐1 (29F.1A12) and rat IgG2a isotype control (2A3) were purchased from Bio X cell.

MaruYama T., Kobayashi S., Shibata H., Chen W, & Owada Y. (2021). Curcumin analog GO‐Y030 boosts the efficacy of anti‐PD‐1 cancer immunotherapy. Cancer Science, 112(12), 4844-4852.

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Antibody Panel for Immune Profiling

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The following antibodies were obtained from eBioscience: CD11b (M1/70)-APC-eFluor 780, CD11c (N418)-PerCP-Cyanine5.5, CD45.1 (A20)-FITC and PE, CD45R/B220 (RA3-6B2)-PE, and Ki-67 (SolA15)-PE-Cyanine7. The following antibodies were obtained from Invitrogen: CD45 (30-F11)-eFluor 450 and GM-CSF (MP1-22E9)-FITC. The following antibodies were obtained from BD Pharmingen: Ly6G (1A8)-PE-Cy7. The following antibodies were obtained from BioLegend: CD4 (RM4-5)-APC. For in vivo GM-CSF blocking experiments, 500 μg of anti–mouse GM-CSF (MP1-22E9) or rat IgG2a isotype control (2A3) (BioXCell) was administered via i.p. injection.

Atkinson J.R., Jerome A.D., Sas A.R., Munie A., Wang C., Ma A., Arnold W.D, & Segal B.M. (2022). Biological aging of CNS-resident cells alters the clinical course and immunopathology of autoimmune demyelinating disease. JCI Insight, 7(12), e158153.

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