Apc rat igg2b

Manufactured by BioLegend

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The APC Rat IgG2b is a laboratory reagent used in flow cytometry applications. It serves as an isotype control to establish background fluorescence levels when analyzing cell samples.

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IgG2b (19 citations) Rat IgG2b (14 citations) Rat IgG2b κ-APC (3 citations) APC‐conjugated rat IgG2b (2 citations)

7 protocols using apc rat igg2b

Multicolor Flow Cytometric Analysis

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Cells were stained with Zombie NIR Fixable Viability dye (catalog no. 423106; BioLegend, San Diego, CA, United States), which is non-permeant to live cells but is permeant to cells with compromised membranes, i.e., dead cells. The cells were then pre-incubated with purified anti-mouse CD16/32 antibody (catalog no. 101310; BioLegend) to block non-specific immunoglobulin binding to the Fc receptors. Finally, the cells were incubated with specific antibodies or isotype controls according to the manufacturers’ guidelines. The antibodies used were: Pacific Blue anti-mouse CD45; allophycocyanin-conjugated (APC) anti-mouse CD3; fluorescein isothiocyanate–conjugated (FITC) anti-mouse CD8a; peridinin chlorophyll protein complex–conjugated (PerCP) anti-mouse CD4; FITC anti-mouse CD19; phycoerythrin-conjugated (PE) anti-mouse CD366; Pacific Blue Rat immunoglobulin (Ig)G2b, κ Isotype Ctrl; APC Rat IgG2b, κ Isotype Ctrl; FITC Rat IgG2a, κ Isotype Ctrl; PerCP Rat IgG2b, κ Isotype Ctrl; and PE Rat IgG2a, κ Isotype Ctrl (all from BioLegend). The cells were detected and analyzed using a FACSAria III flow cytometer (BD Biosciences, San Jose, CA, United States); positive cells were defined using an isotype control or a fluorescence minus one control.

Zhang Y., Jiang N., Zhang T., Wang D., Feng Y., Sang X., Yang N, & Chen Q. (2018). Toxoplasma gondii Genotype Determines Tim-3 Expression Levels in Splenic and Circulatory T Cells in Mice. Frontiers in Microbiology, 9, 2967.

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Isolation and Characterization of Murine Immune Cells

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Chicken egg ovalbumin (OVA, premium quality Grade V) was obtained from Sigma-Aldrich (St. Louis, MO). A TRPV4 KO mouse line on a C57BL/6 background was generated by Dr. Makoto Suzuki (Jichi Medical University, Tochigi, Japan) as described previously [22 (link)]. Inbred C57BL/6 mice (six-eight-week old) were obtained from Charles River (Wilmington, MA). Both male and female WT and TRPV4 KO mice were used in the experiments. The Institutional Animal Care and Use Committee (IACUC) of the University of Maryland reviewed and approved all animal research pertaining to this project, and all the animals were housed in HEPA filtered animal cages and provided ad libitum access to food and water.
APC-Cy7 rat anti-mouse CD45, APC-Cy7 rat IgG2b, FITC hamster anti-mouse CD11c, FITC Ar hamster IgG1, PE rat anti-mouse siglec-F, PE-rat IgG2a, APC-Cy7 rat anti-mouse Ly6G and Ly6C (Gr1), and APC-Cy7 rat IgG2b were purchased from BD Pharmingen (San Diego, CA); APC anti-mouse/human CD11b, APC rat IgG2b, and PE rat anti-mouse F4/80 were purchased from Biolegend (San Diego, CA).

Palaniyandi S., Rajendrakumar A.M., Periasamy S., Goswami R., Tuo W., Zhu X, & Rahaman S.O. (2019). TRPV4 is dispensable for the development of airway allergic asthma. Laboratory investigation; a journal of technical methods and pathology, 100(2), 265-273.

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Multiparametric Flow Cytometry of Immune Cell Populations

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FcR blocking was performed with FcR blocking reagent, mouse (Miltenyi Biotec). Antibodies used were anti-Ly-6B.2-Alexa Fluor 700 (Bio-Rad), anti-CD11b-APC, anti-Ly-6G Pacific Blue, anti-CD3-BV605, or anti-CD138-PerCP/Cy5.5 (BioLegend). Levels of apoptosis were assessed by TUNEL staining using the in situ bromodeoxyuridine (BrdU) DNA fragmentation kit (Abcam) followed by staining with anti-BrdU-APC (eBioscience) and flow cytometric analysis. Isotype controls were used accordingly: Alexa Fluor 700 Rat IgG2a, κ (clone RTK2758), APC Rat IgG2a, κ (clone RTK2758), APC Rat IgG2b, κ (clone RTK4530), BV605 Rat IgG2b, κ (clone 17A2), APC/Cy7 Rat IgG2c, κ (clone RTK4174), Pacific Blue Rat IgG2a, κ (clone RTK2758), PerCP/Cy5.5 Rat IgG2b, κ (clone RTK4530) (BioLegend). AbCTM anti-Rat/Hamster Bead kit (Molecular Probes, Thermo Fisher Scientific) was used for single-color compensation. Flow cytometry data were collected on an LSRII flow cytometer with BD FACSDiva software (BD Biosciences).

Lilly C.L., Villa N.Y., Lemos de Matos A., Ali H.M., Dhillon J.K., Hofland T., Rahman M.M., Chan W., Bogen B., Cogle C, & McFadden G. (2016). Ex Vivo Oncolytic Virotherapy with Myxoma Virus Arms Multiple Allogeneic Bone Marrow Transplant Leukocytes to Enhance Graft versus Tumor. Molecular Therapy Oncolytics, 4, 31-40.

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Immune Cell Quantification in Wounded Tissues

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The cells obtained from the wounded tissues were incubated with Anti-Mouse CD16/CD32 (clone 2.4G2, BD Biosciences, Franklin Lakes, NJ, USA) on ice for 15 min in PBS that contained 1% FCS and 0.1% sodium azide. The cells were stained with Pacific blue-anti-CD45 monoclonal antibody (mAb) (clone 30-F11, BioLegend, San Diego, CA, USA), APC-anti-CD11b mAb (clone M1/70, BioLegend, San Diego, CA, USA), APC/Cy7-anti-Ly6G mAb (clone 1A8, BioLegend, San Diego, CA, USA), and FITC-anti-F4/80 mAb (clone BM8, BioLegend, San Diego, CA, USA). Isotype-matched irrelevant IgG (Pacific: blue Rat IgG2b, κ Isotype Ctrl mAb, APC Rat IgG2b, κ Isotype Ctrl, APC/Cy7 Rat IgG2a, κ Isotype Ctrl, and FITC Rat IgG2a, κ Isotype Ctrl, BioLegend, San Diego, CA, USA) was used for control staining. The stained cells were analyzed using a BD FACS CantoTM II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FACS Diva software. Neutrophils and macrophages were identified as CD45⁺CD11b⁺Ly6G⁺ cells and CD45⁺CD11b⁺F4/80⁺ cells, respectively. The number of neutrophils and macrophages was estimated by multiplying the total leukocyte number by the proportion of each fraction.

Tanno H., Kanno E., Sato S., Asao Y., Shimono M., Kurosaka S., Oikawa Y., Ishi S., Shoji M., Sato K., Kasamatsu J., Miyasaka T., Yamamoto H., Ishii K., Imai Y., Tachi M, & Kawakami K. (2021). Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds. International Journal of Molecular Sciences, 22(8), 3931.

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Flow Cytometry Assay for PD-L1

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To separate the cells, we utilized the FACSCalibur (BD), followed by the FACSAria (BD). Surface staining assays for flow cytometry and cell sorting assays were conducted. Single cells were suspended in 200 µl of PBS (containing 1% serum) with or without labeled antibodies, following the manufacturer’s protocol. The APC-labeled anti-mouse PD-L1 antibody (Cat#124312; Biolegend) and PE-labeled anti-human PD-L1 antibody (Cat#329706; Biolegend) were employed to stain human and murine PD-L1 in flow cytometry sorting. Isotype PE mouse IgG2b (Cat# 400313, Biolegend) and APC rat IgG2b (Cat# 400612, Biolegend) were used as isotype control antibodies.

Gu T., Tian X., Wang Y., Yang W., Li W., Song M., Zhao R., Wang M., Gao Q., Li T., Zhang C., Kundu J.K., Liu K., Dong Z, & Lee M.H. (2023). Repurposing pentamidine for cancer immunotherapy by targeting the PD1/PD-L1 immune checkpoint. Frontiers in Immunology, 14, 1145028.

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Multiparameter Flow Cytometry of Cell Surface Markers

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The following antibodies of cell surface molecules were used: Allophycocyanin (APC) anti-mouse CD45 (Thermo Fisher Scientific Cat# MCD4505, RRID:AB_10376146), anti-mouse CD31 (BioLegend Cat# 102410, RRID:AB_312905), anti-mouse CD166 (Thermo Fisher Scientific Cat# 17-1661-82, RRID:AB_2573170), anti-mouse CD200 (BioLegend Cat# 123810, RRID:AB_10900447), anti-mouse SCA1 (BioLegend Cat# 122511, RRID:AB_756196), anti-mouse CD105 (BioLegend Cat# 120414, RRID:AB_2277914); APC Rat IgG2a, κ isotype control antibody (BioLegend Cat# 400512, RRID:AB_2814702), APC Rat IgG2b, κ isotype control antibody (BioLegend Cat# 400612, RRID:AB_326556), APC Rat IgG1, κ isotype control (BioLegend Cat# 400412, RRID:AB_326518); Alexa Fluor 647 anti-mouse CD9 (BioLegend Cat# 124810, RRID:AB_2076037), Alexa Fluor 647 Rat IgG2a, κ isotype control antibody (BioLegend Cat# 400526, RRID:AB_2864284).
Cells (n=3 with cells from three different mice) were stained with antibodies or IgG isotype controls for 30 min at room temperature. Stained cells were analyzed on a FACSCalibur or BD LSR II flow cytometer (BD Biosciences). Positive cells were gated based on both unstained and isotype-matched IgG-stained cells. Data analysis was performed using FlowJo software (BD Biosciences).

Zhang F., Wang Y., Zhao Y., Wang M., Zhou B., Zhou B, & Ge X. (2023). NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation. eLife, 12, e81569.

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Monoclonal Antibodies for Immune Cell Analyses

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MAb to C3a receptor (C3aR) (cat# sc-133172, clone D-12, IgG2a, κ, 1:50) was purchased from Santa Cruz Biotechnology, Dallas, TX. MAbs against mouse CD45 (cat# 103128, clone 30-F11, rat IgG2b, κ, 1:100), C5a receptor-1 (C5aR1, CD88) (cat# 135808, clone 20/70, rat IgG2b, κ, 1:100), IL-17A (cat# 506908, clone TC11–18H10.1, rat IgG1, κ, 1:100), CD4 (cat# 100412, clone GK1.5, rat IgG2b, κ, 1:100), TCRβ (cat# 109224, clone H57–597, armenian hamster IgG, 1:100), IL-6Rα chain (CD126) (cat# 115815, clone D7715A7, rat IgG2b, κ, 5 μg/mouse), Ly6G (cat# 127616, clone 1A8, rat IgG2a, κ, 1:100), EpCAM (cat# 118208, clone G8.8, rat IgG2a, κ, 1:100), rat IgG2b, κ isotype control (cat# 400644, clone RTK4530, 1:100 or 5 μg/mouse), PE mouse IgG2a, κ isotype control (cat# 400213, clone MOPC-173, 1:50), APC rat IgG2b, κ isotype control (cat# 400611, clone RTK4530, 1:100), Alexa Fluor 700 rat IgG2b, κ isotype control (cat# 400628, clone RTK4530, 1:100), FITC rat IgG2a, κ isotype control (cat# 400505, clone RTK2758, 1:100), were from Biolegend, San Diego, CA. MAbs to EpCAM (cat# 563478, clone G8.8, rat IgG2a, κ, 1:100), IL-6 (cat# 554401, clone MP5–20F3, rat IgG1, 1:100), APC rat IgG2a κ isotype control (cat# 554690, clone R35–95, 1:100) and PE rat IgG1, κ isotype control (cat# 554685, clone R3–34, 1:100) were obtained from BD Bioscience, Franklin Lakes, NJ.

Wang H., Ideguchi H., Kajikawa T., Mastellos D.C., Lambris J.D, & Hajishengallis G. (2022). Complement is required for microbe-driven induction of Th17 and periodontitis. Journal of immunology (Baltimore, Md. : 1950), 209(7), 1370-1378.

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