Rmg1 1

Manufactured by BioLegend

RMG1-1 is a mouse monoclonal antibody that recognizes the Granzyme B protein. Granzyme B is a serine protease found in the cytotoxic granules of natural killer cells and cytotoxic T lymphocytes, and plays a role in inducing apoptosis in target cells.

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Lab products found in correlation

Streptavidin hrp, by BioLegend (1 mentions) Il 17, by BioLegend (1 mentions) Il 13, by Thermo Fisher Scientific (1 mentions) Opteia tmb substrate set, by BD (1 mentions) Clariostar, by BMG Labtech (1 mentions) Ifn γ, by BioLegend (1 mentions) Goat anti rabbit igg hrp, by Agilent Technologies (1 mentions) Anti erp57, by Enzo Life Sciences (1 mentions) Rabbit anti mouse igg hrp, by Agilent Technologies (1 mentions) Streptavidin horseradish peroxidase, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Biotinylated goat anti mouse igg, by Southern Biotech (1 mentions) Igg2c, by Southern Biotech (1 mentions) Igg2b, by BioLegend (1 mentions) Murine il 4, by Thermo Fisher Scientific (1 mentions) Elisa kit, by Thermo Fisher Scientific (1 mentions) Cd43 negative selection, by Miltenyi Biotec (1 mentions) Murine il 7, by Thermo Fisher Scientific (1 mentions) Hi fbs, by Thermo Fisher Scientific (1 mentions) Rme 1, by BioLegend (1 mentions)

6 protocols using rmg1 1

Measuring HDM-specific IgG1 and Cytokines

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Supernatant and BAL cytokine measurements were performed using commercial IL-13 (eBioscience), IL-17 (BioLegend), IFNγ (BioLegend), and IL-5 (BioLegend) ELISA kits. All commercial ELISA assays were performed according to the manufacturer’s supplied instructions. To measure serum HDM-specific IgG1, a 96-well plate (Corning Costar 3361) was first coated overnight at 4°C with 25 mg/ml HDM in PBS. After plate washing, serum samples and controls were added and incubated at 23°C for 2 hours followed by washing and incubation with a detection biotinylated anti-mouse IgG1 (Biolegend, RMG1-1) at 1:750 dilution for 60 minutes. After washing, streptavidin-HRP (Biolegend) was added for 30 minutes. The plate was washed again, and the ELISA was developed with BD OptEIA TMB substrate set (BD Biosciences). OD measurements were recorded at 450–570 nm using a CLARIOstar (BMG Labtech) plate reader. A reference standard for HDM-specific IgG1 ELISAs was generated by pooling serum samples from HDM-sensitized mice, and varied dilutions of this standard were used to generate a standard curve. ELISA results were scaled such that this reference sample has a relative abundance of HDM-specific IgG1 of 1, and the same pooled reference standard was used for all HDM-specific IgG1 ELISAs reported in this manuscript.

Skalski J.H., Limon J.J., Sharma P., Gargus M.D., Nguyen C., Tang J., Coelho A.L., Hogaboam C.M., Crother T.R, & Underhill D.M. (2018). Expansion of commensal fungus Wallemia mellicola in the gastrointestinal mycobiota enhances the severity of allergic airway disease in mice. PLoS Pathogens, 14(9), e1007260.

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Analyzing HLA Protein Expression

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Anti-β₂m (polyclonal, #A0072, Dako), anti-CRT (polyclonal #ABR-01176, Dianova), anti-ERp57 (polyclonal #ADI-SPA-585, Enzo Life Sciences), anti-HLA-A/B/C (W6/32, AbD Serotec®), anti-HLA-A/B/C-PE (W6/32, eBioscience), anti-HLA-G (MEM-G/9, Thermo Fisher Scientific), anti-TAP1 (polyclonal #ADI-CSA-620, Enzo Life Sciences), anti-TPN (polyclonal #ADI-CSA-625 J, Enzo Life Sciences), anti-V5 (MCA1360, ABD Serotec), rabbit anti-mouse IgG-HRP (polyclonal, #P0161, Dako), goat anti-rabbit IgG-HRP (polyclonal, #P0448, Dako), and rat anti-mouse IgG-PE (RMG1-1, Biolegend).

Celik A.A., Simper G.S., Hiemisch W., Blasczyk R, & Bade-Döding C. (2018). HLA-G peptide preferences change in transformed cells: impact on the binding motif. Immunogenetics, 70(8), 485-494.

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Measuring Anti-HPV E7 IgG1 Levels

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Within 5–7 weeks after in utero HPV E7 injection or 4 weeks after adoptive transfer of HPV E7 loaded-fetal MPs, the recipients were subjected to blood sampling for measuring serum anti-E7 IgG₁ levels. ELISA microtiter plates were coated with 100 ng/mL HPV E7 peptides overnight, blocked with 3% bovine serum albumin (BSA, Sigma) in PBS and incubated with 100 µL of diluted samples at room temperature for 60 min. After washing, biotinylated anti-mouse IgG₁ (RMG1-1, BioLegend) was added in each well, followed by streptavidin-horseradish peroxidase (Sigma). The reaction was developed by adding 100 µL NeA-blue tetramethylbenzidine substrate (Clinical Science Products) and stopped with 2 M H₂SO₄. The optical density at 450 nm was measured in an automatic ELISA reader. Serum anti-E7 IgG₁ levels were determined by the standard curve of mouse anti-E7 IgG₁ mAb (ED17, Santa Cruz Biotechnology).

Chen J.C., Ou L.S., Kuo M.L., Tseng L.Y, & Chang H.L. (2020). Fetal exposure to oncoantigen elicited antigen-specific adaptive immunity against tumorigenesis. Journal for Immunotherapy of Cancer, 8(1), e000137.

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Murine Serum Antibody Titer ELISA

Cited 1 time

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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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Differentiation of B Cells from OP9-GFP Precursors

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OP9-GFP cells (12 (link)) were maintained
in MEMα (ThermoFischer) with 20% HI-FBS (Hyclone), glutamine,
β-mercaptoethanol, and penicillin/streptomycin. 1E4 sorted preproB cells
were added to 80–90% confluent OP9-GFP cells in 35 mm dishes in IMDM with
10% HI-FBS, 5 ng/ml murine FL, 1 ng/ml murine IL-7 (Peprotech), and
penicillin/streptomycin. Formation of mature myeloid and B lymphoid cells was
assessed using anti-CD11b-PE and anti-CD19-PerCP-Cy5.5 (1D3), and B cell
precursors were evaluated as done for marrow cells, in both cases gating on the
GFP^- population. Splenocytes were subjected to red blood cell
lysis and CD43-negative selection (Miltenyi). CD43^- cells were
cultured in RPMI with 15% HI-FBS, β-mercaptoethanol,
penicillin/streptomycin, and 20 ng/ml murine IL-4 (Peprotech) with either 10
μg/ml anti-murine CD40 (1C10, Biolegend) antibody or 25 μg/ml
E. coli O55:B5 LPS (Sigma), followed by analysis on day 4
using anti-B220-APC with anti-IgE-PE (RME-1, Biolegend) and anti-IgG1-BV421
(RMG1–1, Biolegend) or anti-IgG2b-PE (RMG2b-1, Biolegend). Serum obtained
by lancing the facial vein was analyzed for IgM, IgG, and IgA after 1:10,000
dilution using ELISA kits per the manufacturer’s instructions
(Invitrogen).

Guo H., Barberi T., Suresh R, & Friedman A.D. (2018). Progression from the Common Lymphoid Progenitor to B/Myeloid preproB and proB Precursors During B Lymphopoiesis Requires C/EBPα. Journal of immunology (Baltimore, Md. : 1950), 201(6), 1692-1704.

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Surface and Intracellular Cytokine Staining

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For surface staining, fluorophore-conjugated mAbs specific for CD4 (RM4-5), CD19 (6D5), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), PD-1 (RMP1-30) and TCRβ (H57-597) were obtained from BioLegend. Ab recognizing TCR Vβ14 (14-2) and CXCR5 (2G8) were from BD PharMingen. Isotype controls for TCR Vβ14, Rat IgM kappa, were from BD PharMingen. Anti-TCR Vβ6 (RR4-7) was from eBioscience. For intracellular cytokine staining, cells were incubated for 4 hr with BD GolgiPlug (1:1000 dilution), 50 ng/m: phorbol 12-myristate 13-acetate, and 1 μM ionomycin in DMEM (HyClone) supplemented with 10% FCS, 1% nonessential amino acids, penicillin, streptomycin, and glutamine at 37°C. Intracellular cytokine staining was performed with Cytofix/Cytoperm (BD PharMingen). Abs recognizing IFN-γ (XMG 1.2), IL-17 (TC11-18H10.1) and IgG1 (RMG1-1) were obtained from BioLegend. Cells were run on an LSRII (BD Biosciences), and analyses were performed with FlowJo (TreeStar) software. Abs recognizing IgG1 were also used for ELISPOT.

Bradley C.P., Teng F., Felix K.M., Sano T., Naskar D., Block K.E., Huang H., Knox K.S., Littman D.R, & Wu H.J. (2017). Segmented Filamentous Bacteria Provoke Lung Autoimmunity by Inducing Gut-Lung Axis Th17 Cells Expressing Dual TCRs. Cell host & microbe, 22(5), 697-704.e4.

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