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Mouse igg2b

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Mouse IgG2b is an immunoglobulin G (IgG) class antibody that is derived from mouse. It is a commonly used isotype control in flow cytometry and other immunoassays.

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Lab products found in correlation

Mouse igg1, by BioLegend (8 mentions) Facscalibur, by BD (4 mentions) High binding assay black plates, by Corning (3 mentions) 96 well plate, by Corning (3 mentions) Rat igg2a, by BioLegend (3 mentions) Clone 523203, by R&D Systems (2 mentions) Anti dec 205 cd205, by Bio-Rad (2 mentions) Rat igg2b, by BioLegend (2 mentions) Mouse igg2a, by BioLegend (2 mentions) Fowjo analysis software, by FlowJo (1 mentions) Fix perm, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Streptavidin pe, by Thermo Fisher Scientific (1 mentions) Attune classic flow cytometer, by Thermo Fisher Scientific (1 mentions) Epcam, by Sony (1 mentions) Mouse igg1κ, by BD (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Cd271, by BD (1 mentions) Safire2 fluorescence plate reader, by Tecan (1 mentions) Streptavidin horseradish peroxidase hrp conjugate, by Thermo Fisher Scientific (1 mentions)

20 protocols using mouse igg2b

1

Flow Cytometry Analysis of MC Receptors

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Flow-cytometry was performed according to routine protocols [30 (link)]. Briefly, MCs were blocked for 15 min at 4 °C with human AB-serum (Biotest, Dreieich, Germany) and incubated with specific antibodies for 30 min at 4 °C. The antibodies were as follows: 0.5 µg/mL of anti-human FcεRIα-FITC (clone AER-37, eBioscience, San Diego, CA, USA), Mouse IgG2b, κ-FITC served as isotype control; 0.15 µg/mL PE-labelled anti-human MRGPRX2 (clone K12H4, Biolegend San Diego, CA, USA), Mouse IgG2b-PE (clone eBWG2b, eBioscience) served as isotype control. After washing, cells were measured on the Facscalibur (BD Biosciences, San Jose, CA, USA) and analyzed with the FowJo analysis software (FlowJo LLC, Ashland, OR, USA). Net mean fluorescence intensity (MFI), i.e., “MFI specific antibody–MFI isotype control” served for receptor quantification. Negative values were set as 0.
Wang Z., Guhl S., Franke K., Artuc M., Zuberbier T, & Babina M. (2019). IL-33 and MRGPRX2-Triggered Activation of Human Skin Mast Cells—Elimination of Receptor Expression on Chronic Exposure, but Reinforced Degranulation on Acute Priming. Cells, 8(4), 341.
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2

Flow cytometric analysis of apoptosis

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Staining was performed after co-incubating target cells (THP-1 and monocytes) and T cells (target:T cell ratio = 2:1) with or without the hemibodies and BiTE constructs directed against CD45 or HLA-A2 respectively for 4 h as indicated. For analysis, surface staining for HLA-A2 was performed first, using anti-HLA-A2-APC (Biolegend, mouse IgG2b, used at 1 μl in 50 μl), followed by fixation and permeabilization of the cells (Fix + Perm, BD Biosciences). Rabbit anti-active Caspase-3-PE (BD Biosciences, clone C92-605, 3 μl in 50 μl) was subsequently added for 30 min. Cells were washed with 1× PBS + 5% human serum (HS, PAA Laboratories) and analyzed on a BD-FACS Canto-II. Stepwise gating was performed by first using a FSC/SSC live gate followed by focusing on different target populations (FCS/HLA-A2 gate), thus evaluating HLA-A2-positive and HLA-A2-negative targets separately for active caspase-3 positivity. Various conditions (titrated hemibody concentrations, various HLA-A2 positive and negative tumor cell lines) were tested in at least 12 independent experiments (including THP-1 cells (9×) and monocytes as bystanders (2×).
Banaszek A., Bumm T.G., Nowotny B., Geis M., Jacob K., Wölfl M., Trebing J., Kucka K., Kouhestani D., Gogishvili T., Krenz B., Lutz J., Rasche L., Hönemann D., Neuweiler H., Heiby J.C., Bargou R.C., Wajant H., Einsele H., Riethmüller G, & Stuhler G. (2019). On-target restoration of a split T cell-engaging antibody for precision immunotherapy. Nature Communications, 10, 5387.
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3

Evaluating IgT+ B Cell Depletion by Anti-trout IgT

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To initially evaluate the IgT+ B cell depletion effect of anti-trout IgT mAbs and TAs in vivo, fish (~2-3 g) were intraperitoneally injected with two different doses (2 or 10 μg) of mouse anti-trout IgT mAbs (clone 41.8; IgG2b) (23 (link)) or 10 μg of mouse IgG2b as isotype control antibody (Biolegend). After 24 hours, half of the fish from each group were further injected with 30 μl of TAs (titer ≥ 1:204,800), whereas the other half were injected with TCs. Seven days after antibody injection, blood leukocytes were obtained and the IgT+ and IgM+ B cell populations were stained and evaluated by flow cytometry as described in the Supplementary Methods. After confirmation that effective IgT+ B cell depletion was achieved with the treatment of 10 μg of anti-trout IgT mAbs and TAs, we used for the rest of experimental procedure 2.5 times the amount of the anti-trout IgT mAb (25 μg/fish) to ensure a consistent and high level of IgT+ B cell depletion in all fish. A time course of IgT+ B cell depletion from blood, head kidney and gill were thereafter performed using the 25 μg/fish dose of anti-trout IgT mAb or its corresponding isotype control, in combination with the subsequent injection of TAs in all groups. Concentration of IgT, IgM and IgD in the serum and gill mucus of these fish were measured as described in the Supplementary Methods.
Xu Z., Takizawa F., Casadei E., Shibasaki Y., Ding Y., Sauters T.J., Yu Y., Salinas I, & Sunyer J.O. (2020). Specialization of mucosal immunoglobulins in pathogen control and microbiota homeostasis occurred early in vertebrate evolution. Science immunology, 5(44), eaay3254.
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4

Characterizing Melanoma Heterogeneity

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Primary tumors, lymph nodes, and lungs were enzymatically digested into single-cell suspensions to characterize melanoma heterogeneity as described above. Erythrocytes were lysed with 1 × ACK buffer (1.5 M NH4Cl, 100 mM NaHCO3, 10 mM EDTA) and washed with PBS. Cells from primary tumors, lymph nodes, lungs, and blood were stained for viability using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (L34957, Invitrogen™, Carlsbad, CA) for 20 min at 4 °C. After washing with PBS, the cells were stained with a cocktail of primary antibodies or corresponding isotype controls, including EpCAM (2221100, SONY, Tokyo, Japan), Trop2 (1B-898-C100, Exbio, Vestec, Czech Republic), and CD271 (562122, BD Biosciences, San Jose, CA) (Supplementary Tables 1 and 2), as well as mouse IgG2b (400342, Biolegend, San Diego, CA), and mouse IgG1 κ (557872, BD Biosciences, San Jose, CA). After 20 min of incubation at 4 °C, staining with Streptavidin PE (12-4317, eBioscience, San Diego, CA) was performed for 20 min at 4 °C. The gating of positive populations was done using isotype controls. Only viable single GFP+ cells without debris were included in the analysis. The samples were measured on an Attune Classic flow cytometer (ThermoFisher Scientific, Waltham, MA) and analyzed using FlowJo software.
Pícková M., Kahounová Z., Radaszkiewicz T., Procházková J., Fedr R., Nosková M., Radaszkiewicz K.A., Ovesná P., Bryja V, & Souček K. (2024). Orthotopic model for the analysis of melanoma circulating tumor cells. Scientific Reports, 14, 7827.
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5

Quantifying Amyloid-Beta Levels via ELISA

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The cells were cultured on 96-well plates (Corning) overnight then transfected with pcDNA-TTR or control plasmid. The culture medium was collected after 24 hours. The medium was analyzed by an Aβ ELISA as follows. Monoclonal 6E10 anti-Aβ(residues 1–17) mouse IgG1, (Biolegend) was coated in 50 mm carbonate buffer, pH 9.6, at 4°C overnight on high binding assay black plates (Costar), washed with TBST (tris buffered saline with 0.05% Tween 20) and blocked with 5% non-fat milk in TBST. Samples and standards (synthetic Aβ1-40) were incubated for 2 hr, followed by addition of biotin-labeled 4G8 [anti-Aβ residues 17–24, mouse IgG2b (Biolegend)] and incubation for 1 hr at 37°C. After washing, streptavidin horseradish peroxidase (HRP) conjugate (Invitrogen) was added and incubated for 45 min, followed by detection by Quanta Blue fluorogenic peroxidase substrate (Thermo Scientific) using a Tecan Safire II fluorescence plate reader.
Li X., Song Y., Sanders C.R, & Buxbaum J.N. (2016). Transthyretin Suppresses Amyloid-β secretion by Interfering with Processing of The Amyloid-β Precursor Protein. Journal of Alzheimer's disease : JAD, 52(4), 1263-1275.
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6

Quantitative Analysis of LPA1 Expression

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FLSs were stained with anti-LPA1 monoclonal antibody (mAb) (1G6; LSBio, Seattle, WA, USA) as a first antibody, and phycoerythrin-conjugated anti-mouse immunoglobulin G (IgG) antibody (BioLegend, San Diego, CA, USA) as a second antibody. Mouse IgG2b (BioLegend) was used as an isotype control. Cells were then analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).
Miyabe Y., Miyabe C., Iwai Y., Yokoyama W., Sekine C., Sugimoto K., Harigai M., Miyasaka M., Miyasaka N, & Nanki T. (2014). Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade. Arthritis Research & Therapy, 16(5), 461.
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7

Isolation and Characterization of Human Skin Dendritic Cells

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Clinically normal appearing skin was derived from plastic surgery for breast or abdominal skin reduction after written patient consent. Ethical approval was granted by the local ethical committee (AN3694 – 279/4.3). Skin samples were trimmed off subcutaneous fat with a scalpel, and 8 mm punch biopsies (Kai Europe, Solingen, Germany) were prepared. The following mAbs were used for targeting DC in human skin: anti-DEC-205/CD205 (five different batches of clone MG38, Serotec, Kidlington, UK; and clone 523203 from R&D Systems, Minneapolis, MN, USA), anti-Langerin/CD207 (clone DCGM4/122D5.03, Dendritics, Lyon, France). Appropriately matched isotype controls (mouse IgG2b and mouse IgG1, respectively, from BioLegend, San Diego, CA, USA) were used.
Stoitzner P., Schaffenrath S., Tripp C.H., Reider D., Komenda K., Del Frari B., Djedovic G., Ebner S, & Romani N. (2014). Human skin dendritic cells can be targeted in situ by intradermal injection of antibodies against lectin receptors. Experimental Dermatology, 23(12), 909-915.
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8

Quantifying Amyloid-Beta Secretion

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7PA2 or 7WD10 cells were cultured on 96-well plates (Corning) and treated with IRE1 activating compounds +/− 4μ8c overnight. The medium was then replaced with fresh medium containing treatment at a reduced volume (50%), culture medium was collected after 24 hrs. The medium was analyzed by an Aβ ELISA as follows. Monoclonal 6E10 anti-Aβ(residues 1–17) mouse IgG1, (Biolegend) was coated in 50 mm carbonate buffer, pH 9.6, at 4°C overnight on high binding assay black plates (Costar), washed with TBST (tris buffered saline with 0.05% Tween 20) and blocked with 5% non-fat milk in TBST. Samples and standards (condition 7PA2 media) were incubated for 1.5 hrs, followed by addition of 4G8 antibody [anti-Aβ residues 17–24, mouse IgG2b (Biolegend)] conjugated to horseradish peroxidase (HRP) and incubated for 1.5 hrs at 25°C. After washing, ABTS substrate was added, followed by detection with an absorbance plate reader.
Grandjean J.M., Madhavan A., Cech L., Seguinot B.O., Paxman R.J., Smith E., Scampavia L., Powers E.T., Cooley C.B., Plate L., Spicer T.P., Kelly J.W, & Wiseman R.L. (2020). Pharmacologic IRE1/XBP1s Activation Confers Targeted ER Proteostasis Reprogramming. Nature chemical biology, 16(10), 1052-1061.
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9

PD-L1 and PD-L2 Expression Analysis

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Cells were stained for PE-conjugated anti-human PD-L2 (Cat#329606, Biolegend) or APC-conjugated anti-human PD-L1 antibody (Cat#329708, Biolegend) at 1:100 dilution on ice for 30 mins and analyzed by BD FACSCalibur. Isotype controls are mouse IgG2a, κ (Cat#400213, Biolegend) and mouse IgG2b, κ (Cat#400322, Biolegend), respectively. GL261 cells overexpressing PD-L1 and/or PD-L2 were stained for PE-conjugated anti-mouse PD-L2 (Cat#107205, Biolegend) and/or APC-conjugated anti-mouse PD-L1 antibody (Cat#124312, Biolegend) at 1:100 dilution on ice for 30 minutes and sorted using a BD FACSAria III cell sorter. Isotype controls were Rat IgG2a, κ (Cat#400508, Biolegend) and Rat IgG2b, κ (Cat#400612, Biolegend), respectively.
Fu Y., Liu C.J., Kobayashi D.K., Johanns T.M., Bowman-Kirigin J.A., Schaettler M.O., Mao D.D., Bender D., Kelley D.G., Uppaluri R., Bi W.L., Dunn I.F., Tao Y., Luo J., Kim A.H, & Dunn G.P. (2020). GATA2 Regulates Constitutive PD-L1 and PD-L2 Expression in Brain Tumors. Scientific Reports, 10, 9027.
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10

In vivo anti-CD3 and anti-SLAMF4 treatment

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Purified αCD3ε (145-2C11) hamster IgG (Biolegend, San Diego, CA), αSLAMF4 Mouse IgG2b (hybridoma donated by Dr. Vinay Kumar, Un. Chicago or purchased from Biolegend) or Mouse IgG2b (Biolegend, San Diego, CA) were injected i.p., as described 21 (link). Briefly, 20μg of antibody was injected 1, 2, or 3 times at 48 h intervals with the last injection occurring 4 h prior to euthanizing the mice. Slamf4−/− mice were euthanized 16 hours after the second injection.
O’Keeffe M.S., Song J.H., Liao G., De Calisto J., Halibozek P.J., Mora J.R., Bhan A.K., Wang N., Reinecker H.C, & Terhorst C. (2015). SLAMF4 is a Negative Regulator of Expansion of Cytotoxic Intra-epithelial CD8+ T Cells That Maintains Homeostasis in the Small Intestine. Gastroenterology, 148(5), 991-1001.e4.
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