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Igg2b

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IgG2b is an immunoglobulin subclass that functions as an antibody. It is commonly used in various immunological research applications.

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Lab products found in correlation

Igg2a, by BioLegend (4 mentions) Lipopolysaccharides (lps), by Merck Group (3 mentions) Mouse igg1, by BioLegend (2 mentions) Perm buffer, by Thermo Fisher Scientific (1 mentions) Jes5 2a5, by BioLegend (1 mentions) Facsaria flow cytometer, by BioLegend (1 mentions) Na pyruvate, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Monensin, by BioLegend (1 mentions) Incucyte live cell analysis system, by Sartorius (1 mentions) Caspase 3 7 reagent, by Sartorius (1 mentions) Celltrace far red, by Thermo Fisher Scientific (1 mentions) I a 1 e m5 114, by BioLegend (1 mentions) Heat inactivated fcs, by Thermo Fisher Scientific (1 mentions) Foxp3 transcription factor staining buffer kit, by Cytek Biosciences (1 mentions) Zombie aqua fixable viability kit, by BioLegend (1 mentions)

Spelling variants of this product

Rat IgG2b κ isotype control (4 citations) PE Rat IgG2b (4 citations) Mouse IgG2b (MPC-11) (3 citations) PE/Cyanine7 Rat IgG2b (3 citations) Mouse-IgG2b-FITC (2 citations) PE Rat IgG2b (κ) (2 citations) APC‐conjugated rat IgG2b (2 citations) Rat anti-trinitrophenol+KLH IgG2b isotype control (2 citations) PE mouse IgG2b κ (2 citations) Biotinylated rat IgG2b (2 citations)

19 protocols using igg2b

1

Intracellular Cytokine Staining of Tumor-associated B Cells

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Spleen cells from mice bearing active BCL1 tumor cells and naïve mice were suspended at 1 x 106 /mL in stimulation medium prepared using RPMI-1640 (Sigma), L-glutamine (Sigma), NEAA (Gibco), Na-pyruvate (Gibco), 10% heat-inactivated FBS β-mercaptoethanol (50 μM; Sigma), LPS (10 μg/mL; Sigma), PMA (Sigma), Ionomycin (500 ng/mL; Sigma), and monensin (2 μM; BioLegend) and incubated for 5 hours at 37°C, 5% CO2. Samples were washed with cold PBS+10%FBS and stained with antibodies against BCL1-Id, CD3 (145-2C11), CD11b (M1/70), Ter119 (TER119) [for tumor cells] and anti-CD19 (6D5) [normal B cells]. Cells were fixed and permeabilized (Perm Buffer; eBioscience) then stained with anti-IL-10 antibody [JES5-2A5] or isotype control (IgG2b; BioLegend) and analyzed on a FACSAria flow cytometer.
BitMansour A., Pop L.M, & Vitetta E.S. (2016). The Role of Regulatory B Cell-Like Malignant Cells and Treg Cells in the Mouse Model of BCL1 Tumor Dormancy. PLoS ONE, 11(12), e0167618.
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2

NK Cell-Mediated Cytotoxicity Assay

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Target cells (K-562 or P815; see note below) were stained with CellTrace Far Red (Thermo Fisher Scientific) according to the manufacturer’s protocol and immobilized on poly-l-ornithine-coated (MilliporeSigma) clear 96-well flat-bottom plates (1 × 104 cells/well). NK cells were added to each well (2 × 104) for an E/T ratio of 2:1. All cocultures were performed in B0 media supplemented with caspase-3/7 reagent (Essen BioScience). The viable target cells per well were imaged hourly over 24 hours using the IncuCyte Live Cell Analysis System (Essen BioScience). Live cell numbers were quantified using IncuCyte Zoom or S3 software and normalized to the number of live cells in the target only control group. Note: Following staining, but before plating, P815 cells were incubated for 30 minutes at room temperature in B0 media containing either anti-NKp30 (1 μg/mL; BioLegend [catalog 325223]) and isotype (1 μg/mL; R&D Systems [catalog MAB004]), anti-NKp30, and anti-NKG2A (1 μg/mL; Beckman Coulter [catalog IM2750]), or isotype alone (1 μg/mL IgG2b plus 1 μg/mL IgG1; BioLegend [catalog 400124]). Following incubation, cells were washed with PBS and plated as described above.
Myers J.A., Schirm D., Bendzick L., Hopps R., Selleck C., Hinderlie P., Felices M, & Miller J.S. (2022). Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion. JCI Insight, 7(15), e150079.
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3

Immune cell antibody staining

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Mouse antibodies (Abs) and isotype control Abs (IgG1, IgG2a, and IgG2b), anti-CD3 (17A2), CD4 (GK1.5), CD8α (53–6.7), tumor necrosis factor (TNF)-α (MP6-XT22), perforin (S16009A), CD11c (N418), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), anti-interferon (IFN)-γ (B27), MHC class I (H2Kd, 28-8-6), and MHC class II (I-A/I-E, M5/114.15.2) were purchased from BioLegend (San Diego, CA, USA).
Hwang J., Zhang W., Park H.B., Yadav D., Jeon Y.H, & Jin J.O. (2021). Escherichia coli adhesin protein-conjugated thermal responsive hybrid nanoparticles for photothermal and immunotherapy against cancer and its metastasis. Journal for Immunotherapy of Cancer, 9(7), e002666.
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4

Comprehensive Immune Cell Profiling

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Briefly, cells were incubated with an Fc-blocking antibody (clone 2.4G2) at 4° C for 15 min, prior to surface staining. Antibodies to IgA (eBioscience; clone mA-6E1), IgG1 (BioLegend; clone RMG11), IgG2b (BioLegend; clone RMG2b-1), CD4 (BioLegend; clone GK1.5), CD8α (BioLegend; clone 53–6.7), CD19 (BioLegend; clone 6D5), B220 (BioLegend; clone RA3–6B2), TCRβ (BioLegend; clone H57–597), CD25 (BioLegend; clone 3C5) were used with viability determined using a Zombie Aqua™ Fixable Viability Kit (Biolegend). Treg cell staining was conducted using Tonbo Biosciences buffers (Foxp3/Transcription Factor Staining Buffer Kit; cat no: TNB0607-KIT). Cells were stained as above, then fixed for 1 h at RT prior to permeabilzation. The cells were then incubated with the 2.4G2 Fc-blocking antibody at 4° C for 15 min prior to staining with antiFoxP3 PE (eBioscience; clone NRRF-30) for 30 min at 4° C. Hybridoma supernatants containing mAbs used for cell purification or stimulation, were generously provided by the late Charles Janeway Jr. (Yale University). RPMI-1640 media and heat-inactivated FCS were purchased from Invitrogen and Gemini, respectively.
Li Y.Y., Pearson J.A., Chao C., Peng J., Zhang X., Zhou Z., Liu Y., Wong F.S, & Wen L. (2017). Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) modulates T1DM susceptibility by gut microbiota. Journal of autoimmunity, 82, 85-95.
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5

Comprehensive Immune Profiling of Human Brain

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In all, 8*105 single cells from fresh human brain material and cerebral organoids were stained in a 96-well V-bottom plate using the following antibodies from Ebioscience (San Diego, CA): CD45 (#119459), CD11b (#12011841), CD14 (clone 61D3, #90170149025), CD11c (clone 3.9, #11011641), HLA-DR (clone LN3, #17995641), CX3CR1 (clone eB149/10H5, #17609941), IgG1 (#114714 and #17471441), IgG2b (#17403181); antibodies from BD Bioscience: CD206 (#561763); antibodies from BioLegend IgG2b (#400319). Protein expression was quantified on a FACSCanto II (BD Biosciences) and analyzed with FACSDiva software (Version 8.0.1, BD Biosciences). Protein expression was measured in cells that were gated to be alive, single, and CD11b+ (Supplementary Fig. 5b). The geoMFI mean was subtracted from the geoMFI of the corresponding isotype control.
Ormel P.R., Vieira de Sá R., van Bodegraven E.J., Karst H., Harschnitz O., Sneeboer M.A., Johansen L.E., van Dijk R.E., Scheefhals N., Berdenis van Berlekom A., Ribes Martínez E., Kling S., MacGillavry H.D., van den Berg L.H., Kahn R.S., Hol E.M., de Witte L.D, & Pasterkamp R.J. (2018). Microglia innately develop within cerebral organoids. Nature Communications, 9, 4167.
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6

B Cell Class Switch Recombination Assay

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For primary B cells, CSR was initiated by adding 20 μg/ml LPS (Sigma) and 20 ng/ml IL-4 (R&D Systems) to IgG1, 20 μg/ml LPS (Sigma) to IgG3, and 10 μg/ml LPS (Sigma), 2 ng/ml TGF-beta (R&D Systems) and 0.33 μg/ml anti-IgD dextran (Fina Biosolutions) to IgG2b for 72 h (or 48 h if the inversion-deletion reaction was taking place in the presence of LPS for 24 h). For CH12–F3 cells, CSR to IgA was initiated by adding 20 μg/ml LPS, 20 ng/ml IL-4, and 1 ng/ml TGF-beta. After culture, cells were placed in 2.5% FBS in 1X PBS, stained with the appropriate conjugated antibodies (FITC-conjugated anti-IgA (BD Bioscience), IgG1 (BD Bioscience), IgG3 (BD Bioscience), CD19 (Biolegend), B220 (Bioscience), and PE-conjugated anti-IgG1 (BD Bioscience) and IgG2b (Biolegend)). Data were acquired on a FACSAria cell-sorter and Accuri (BD Biosciences) and FlowJo software was used for analysis.
Kazadi D., Lim J., Rothschild G., Grinstein V., Laffleur B., Becherel O., Lavin M.J, & Basu U. (2020). Effects of senataxin and RNA exosome on B-cell chromosomal integrity. Heliyon, 6(3), e03442.
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7

Monocyte Depletion in Mouse TBI Model

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All mice weighed between 25g–30g. For pan depletion of monocytes, 24 hours prior to TBI or sham injury, C57BL/6 male mice underwent monocyte or sham depletion via intravenous injection of liposome encapsulated clodronate or liposome encapsulated PBS as control (Liposoma, Amsterdam). Liposomes were administered according to the recommended dose of 10uL/1g of mouse. For selective depletion of Ly6Chi monocytes, C57BL/6 mice were given intravenous injections of an anti-CCR2 monoclonal antibody (clone: MC21) 24hrs prior to TBI at a dose of 25ug/mouse. The antibody was generated and characterized by Mack et al. as described in (23 (link)) and gifted to our lab by Steffen Jung (19 (link)). The isotype control rat IgG2b (Biolegend) was administered at the same dose. Peripheral blood was assessed before injection to confirm the presence of monocytes. 24 hours post injury the animals were euthanized, blood was collected via cardiac puncture and brains were collected and processed as described above. Leukocyte subsets were identified and counted via flow cytometry as described above. Data was analyzed with the statistical software program PRISM and are reported as the mean ± SEM.
Makinde H.M., Cuda C.M., Just T.B., Perlman H.R, & Schwulst S.J. (2017). Non-classical monocytes mediate secondary injury, neurocognitive outcome, and neutrophil infiltration after Traumatic Brain Injury. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3583-3591.
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8

Multiparametric Flow Cytometry Analysis

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Cell preparations from spleen, thymus, lymph nodes, liver, epididymal fat pads, lung, or Peyer's patches were harvested and exposed to hypotonic lysis to erythrocytes. Following cell preparation, cells were stained and analyzed using a BD LSRFortessa and a Sony Spectral Flow Cytometer. CD1d‐PBS57 (CD1d‐αgal) tetramers were obtained from the NIH Tetramer Core Facility. The following antibodies used for staining were obtained from BioLegend: IFNγ (Clone XMG1.2, Cat 505830), IL‐4 (Clone 11B11, Cat 504109), T‐bet (Clone 4B10, Cat 644816), CD3ε (Clone 17A2, Cat 100241), GL7 (Clone GL7, Cat 144609), B220 (Clone RA3‐6B2, Cat 103243), IgG1 (Clone RMG1‐1, Cat 406610), IgG2b (Clone RMG2b‐1, Cat 406707), and IgD (Clone 11‐26c.2a, Cat 405711). The following antibodies were from eBioscience: RORγt (Clone B2D, Cat 17‐6981‐80) and PLZF (Clone Mags.21F7, Cat 53‐9320‐82). The following antibody is from BD Pharmingen: IgA (Clone C10‐3, Cat 559354).
Clancy‐Thompson E., Chen G.Z., LaMarche N.M., Ali L.R., Jeong H., Crowley S.J., Boelaars K., Brenner M.B., Lynch L, & Dougan S.K. (2019). Transnuclear mice reveal Peyer's patch iNKT cells that regulate B‐cell class switching to IgG1. The EMBO Journal, 38(14), e101260.
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9

Murine Serum Antibody Titer ELISA

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To determine antibody titers in murine serum, HiBond ELISA plates were coated with whole GAS-M1 bacteria, or recombinant M1 protein or isolated M1 HVR [generated as described in (38 (link))] and incubated with serially diluted serum. Captured antibodies were detected using biotinylated goat anti-mouse IgG (Southern Biotech, CAT 1036-08), IgG1 (BioLegend, RMG1-1), IgG2b (BioLegend, RMG2b-1), and IgG2c (Southern Biotech, CAT 1079-08) and streptavidin-HPR (BioLegend, CAT 405210). BSA-coated wells served as negative controls and presented values represent the average of duplicate experimental values with deducted negative control values. All samples were run in duplicate.
Emami S., Rojas Converso T., Persson J.J, & Johansson-Lindbom B. (2023). Insertion of an immunodominant T helper cell epitope within the Group A Streptococcus M protein promotes an IFN-γ-dependent shift from a non-protective to a protective immune response. Frontiers in Immunology, 14, 1241485.
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10

RSV F-specific Antibody ELISA

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RSV F-specific IgG antibodies were assessed using standard ELISA techniques. High binding 96 well plates were coated with purified RSV sF. After blocking, serial dilutions of serum were added to plates. For mouse, bound antibodies were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a (Jackson ImmunoResearch, West Grove, PA) and developed with 3,3´,5,5´-tetramethylbenzidine (TMB, Sigma, St. Louis, MO). For rat, bound antibodies were detected using biotin-conjugated goat anti-rat IgG1, IgG2a, or IgG2b (Biolegend, San Diego, CA) followed by streptavidin-HRP (BD Biosciences, San Jose, CA) and developed with TMB. Absorbance was measured at 450 nm on a SpectraMax plate reader and analyzed using SoftMax Pro (Molecular Devices, Sunnyvale, CA). Titers are reported as log2 endpoint titers using a cutoff of 3x the mean of the blank wells.
Lambert S.L., Aslam S., Stillman E., MacPhail M., Nelson C., Ro B., Sweetwood R., Lei Y.M., Woo J.C, & Tang R.S. (2015). A Novel Respiratory Syncytial Virus (RSV) F Subunit Vaccine Adjuvanted with GLA-SE Elicits Robust Protective TH1-Type Humoral and Cellular Immunity In Rodent Models. PLoS ONE, 10(3), e0119509.
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