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Igg2a isotype control

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The IgG2a isotype control is a laboratory reagent used in flow cytometry and other immunoassays. It serves as a non-specific control to establish baseline signals and differentiate specific from non-specific antibody binding.

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Lab products found in correlation

Sytox green, by Thermo Fisher Scientific (3 mentions) Il 12, by Miltenyi Biotec (2 mentions) Brefeldin a, by Thermo Fisher Scientific (2 mentions) Il 18, by R&D Systems (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Anti mr1 26, by BioLegend (1 mentions) Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Etoposide, by Abcam (1 mentions) Sybr gold, by Thermo Fisher Scientific (1 mentions) Hydrogen peroxide solution, by Merck Group (1 mentions) Yoyo 1, by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions) Alternaria alternata, by Stallergenes Greer (1 mentions) L glutathione gsh, by Merck Group (1 mentions) Decanoyl rvkr cmk, by Bio-Techne (1 mentions) N acetyl l cysteine, by Merck Group (1 mentions) Pamam dendrimer, by Merck Group (1 mentions)

4 protocols using igg2a isotype control

1

MAIT Cell Activation Assay with MR1

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A total of 100,000 HeLa cells stably transfected with MR1 (HeLa‐MR122) were cultured in Dulbecco's modified Eagle's medium (Gibco) without supplement at 37°C, with Escherichia coli (DH5α strain) diluted to reach multiplicities of infection of 1000 (1000 multiplicity of infection), as previously described.22 The PBMCs (5 × 105 per well, in 48‐well plates) were added for 20 hours, and monensin was added for the last 4 hours of culture. For blocking conditions, anti‐MR1 (26.5, Biolegend) or IgG2a isotype control (BD Biosciences) was added at a 10‐μg/mL final concentration. Activation of MAIT cells was measured by flow cytometry using CD3, CD4, CD8, CD161, TCR Vα7.2, CD69, GrB, interferon gamma (IFN‐γ), and LIVE/DEAD Fixable Aqua stain (Invitrogen).
Renand A., Habes S., Mosnier J., Aublé H., Judor J., Vince N., Hulin P., Nedellec S., Métairie S., Archambeaud I., Brouard S., Gournay J, & Conchon S. (2018). Immune Alterations in Patients With Type 1 Autoimmune Hepatitis Persist Upon Standard Immunosuppressive Treatment. Hepatology Communications, 2(8), 968-981.
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2

PBMC and CD8+ T cell activation

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Peripheral blood mononuclear cells (PBMCs) were obtained from adults (whole
blood leukocyte cones, NHS Blood and Transplant, UK), 24 month olds (prospective birth
cohort (Saghafian-Hedengren et al., 2010 (link))) and
umbilical cord blood samples (Stem Cell Services, NHS Blood and Transplant, UK). These
were rested overnight or stored in liquid nitrogen until required. THP-1 cells (ECACC, UK)
were incubated with paraformaldehyde-fixed E. coli (DH5α,
Invitrogen) at 25 bacteria per cell overnight. PMBCs (24 month donors) or sort-purified
CD8+ T cells (adults, sorted on a MoFlo, Beckman Coulter) were then added for a 5h
co-culture. Anti-MR1 antibody (clone 26.5, kindly provided by T.H. Hansen) or IgG2a
isotype control (BD Bioscience) was added at 10µg/ml. For IL-12 + IL-18
stimulations, IL-12 (Miltenyi Biotec) and IL-18 (R&D Systems) were added at
50ng/ml overnight. In all cases, brefeldin A (eBioscience) was added for the final 4hrs of
incubation.
Fergusson J.R., Smith K.E., Fleming V.M., Rajoriya N., Newell E.W., Simmons R., Marchi E., Björkander S., Kang Y.H., Swadling L., Kurioka A., Sahgal N., Lockstone H., Baban D., Freeman G.J., Sverremark-Ekström E., Davis M.M., Davenport M.P., Venturi V., Ussher J.E., Willberg C.B, & Klenerman P. (2014). CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages. Cell Reports, 9(3), 1075-1088.
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3

Analyzing Allergen-Induced Cellular Responses

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Alternaria alternata and house dust mite (HDM; Dermatophagoides farina) extracts were obtained from Greer Laboratories (Lenoir, NC). Hydrogen peroxide solution, l-glutathione (GSH), N-acetyl-l-cysteine (NAC), PAMAM dendrimer, and mouse genomic DNA were purchased from Sigma-Aldrich (St Louis, Mo). AZ 10417808 (AQZ-1), PD 150606, and decanoyl-RVKR-CMK were procured from Tocris Bioscience (Bristol, United Kingdom). Hanks balanced salt solution (HBSS), acetoxymethyl ester form of fura-2 (fura-2 AM), SYBR Gold, YoYo-1, SYTOX Green, 4′,6-diamidino-2-phenylindole (DAPI), and EDTA were obtained from Thermo Fisher Scientific (Waltham, Mass). Etoposide was acquired from Abcam (Cambridge, United Kingdom). Naphthofluroscein, caspase-3, and calpain small interfering RNA (siRNA), control siRNA (fluorescein conjugate), siRNA transfection reagent, and media were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Fluorescently labeled antibodies to CD3 (145-2C11), CD25 (PC51), CD44 (IM7), CD16/CD32 (2.4G2), CD14 (rmC5-3), CD45R/B220 (RA3-6B2), and IgG2a isotype control were purchased from BD Biosciences (San Jose, Calif).
Lee C.G., Soares V.D., Newberger C., Manova K., Lacy E, & Hurwitz J. (1998). RNA helicase A is essential for normal gastrulation. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13709-13713.
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4

Activation of MAIT cells by E. coli

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PBMCs were obtained from adults (whole blood leukocyte cones; NHS Blood and Transplant), 24 month olds (prospective birth cohort; Saghafian-Hedengren et al., 2010 (link)), and umbilical cord blood samples (Stem Cell Services, NHS Blood and Transplant) after appropriate ethical review. These were rested overnight or stored in liquid nitrogen until required.
THP-1 cells (ECACC) were incubated with paraformaldehyde-fixed E. coli (DH5α; Invitrogen) at 25 bacteria per cell overnight. PMBCs (24-month donors) or sort-purified CD8+ T cells (adults; sorted on a MoFlo; Beckman Coulter Genomics) were then added for a 5 hr coculture. Anti-MR1 (clone 26.5, kindly provided by T.H. Hansen) or IgG2a isotype control (BD Biosciences) was added at 10 μg/ml. For IL-12 + IL-18 stimulations, IL-12 (Miltenyi Biotec) and IL-18 (R&D Systems) were added at 50 ng/ml overnight. In all cases, brefeldin A (eBioscience) was added for the final 4 hr of incubation.
Fergusson J.R., Smith K.E., Fleming V.M., Rajoriya N., Newell E.W., Simmons R., Marchi E., Björkander S., Kang Y.H., Swadling L., Kurioka A., Sahgal N., Lockstone H., Baban D., Freeman G.J., Sverremark-Ekström E., Davis M.M., Davenport M.P., Venturi V., Ussher J.E., Willberg C.B, & Klenerman P. (2014). CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages. Cell Reports, 9(3), 1075-1088.
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