Igg isotype control

Manufactured by BD

Sourced in Australia, United States

The IgG isotype control is a laboratory reagent used to determine the specificity of antibody binding in immunoassays. It serves as a negative control, providing a reference point to distinguish specific antibody binding from non-specific background signals.

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Lab products found in correlation

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Spelling variants of this product

Isotype controls (87 citations) Control IgG (13 citations) Isotype IgG (5 citations) PE-conjugated mouse IgG isotype control (4 citations) Mouse IgG isotype control (3 citations) Isotypes (2 citations) Appropriate antibody IgG isotype controls (2 citations) IgG and IgM isotype controls (2 citations) Fluorochrome-conjugated mouse IgG isotype control (2 citations) Rat-IgG isotype control (2 citations)

34 protocols using igg isotype control

Comprehensive Immunophenotyping and Cell Cycle Analysis of MSCs and MSC-MVs

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MSCs were collected for immunophenotypic analysis at P5, 14, and 22. Phycoerythrin (PE)-labeled anti-CD29, CD44, CD73, CD90, CD105, CD34, CD45 and IgG isotype control (all from BD Biosciences, San Diego, CA, USA) were incubated with MSCs for 30 minutes at 4 ºC. After washing in PBS for three times, MSCs were analyzed by flow cytometry (BD Biosciences).
To determine the immunophenotypic profile of MSC-MVs, 1 μm of standard microbeads (Sigma Aldrich, St. Louis, MO) were added to the MSC-MV samples. All samples were stained with Calcein-AM (Sigma), which was used to define the MSC-MVs with intact membrane structure. Subsequently, phycoerythrin (PE)-labeled anti-CD29, CD44, CD73, CD90, CD105, CD34, CD45 and IgG isotype control (BD Biosciences) were incubated with MSC-MV samples for 30 min at 4ºC. After washing in PBS for three times, MSC-MV samples were analyzed by FCM.
For cell cycle analysis, MSCs were trypsinized, centrifuged and washed for three times in PBS. Cells were then resuspended and fixed in 70% ethanol at 4ºC overnight. Fixed cells were washed at 500g for 5 min and stained with 10 μg/ml PI, 100 μg/ml DNase-free RNase A, 0.1% Triton X-100 and kept in the dark for 30 min. Cell suspensions were filtered and measured by FCM.

Lei Q., Liu T., Gao F., Xie H., Sun L., Zhao A., Ren W., Guo H., Zhang L., Wang H., Chen Z., Guo A.Y, & Li Q. (2017). Microvesicles as Potential Biomarkers for the Identification of Senescence in Human Mesenchymal Stem Cells. Theranostics, 7(10), 2673-2689.

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Fluorophore-Conjugated Antibody Staining

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Fluorophores conjugated antibodies were purchased from BD Bioscience (Franklin Lakes, NJ, USA) and DAKO (Agilent Technologies, Santa Clara, CA, USA). The anti-human P2X7 monoclonal antibodies were produced from the L4 clone in house [29 (link)] and conjugated with Alexa 647 or Alexa 488 using the antibody conjugation kit from Molecule Probes (Thermo Fisher Scientific, Waltham, MA, USA).
Cell surface staining was carried out following the BD standard protocol. Aliquots of 100 µL of fresh whole blood were added into fluorescence-activated cell sorting (FACS^®) tubes with pre-mixed antibody cocktails. An autofluorescence tube containing only whole blood and an IgG isotype control (BD Australia, Macquarie Park, NSW, Australia) tube were prepared for each AIBL sample. Titration of each antibody was determined by saturation tests. Blood/antibody mixture was incubated for 15 min at room temperature with gentle shake, followed by incubating with 2 mL of BD FACS Lysing solution (Cat#555899) for another 15 min. 2 mL of PBS was added to each FACS^® tubes, followed by centrifuging at 1400 rpm for 3 min. Supernatant was discarded and leukocytes were resuspended into 200 uL of PBS. Leukocytes were then analysed using FACSCalibur^TM (BD Biosciences) and flow results were primarily analysed using FlowJo software (V10, FlowJo, LLC, Ashland, OR, USA).

Li Y., Huang X., Fowler C., Lim Y.Y., Laws S.M., Faux N., Doecke J.D., Trounson B., Pertile K., Rumble R., Doré V., Villemagne V.L., Rowe C.C., Wiley J.S., Maruff P., Masters C.L, & Gu B.J. (2022). Identification of Leukocyte Surface P2X7 as a Biomarker Associated with Alzheimer’s Disease. International Journal of Molecular Sciences, 23(14), 7867.

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Immunophenotyping of Mouse T Cells

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HEL was purchased from Sigma-Aldrich. The following reagents were from BD Biosciences: 7-AAD, PE-anti-CD4, IgG isotype control, PE-anti-CD25, PE-anti-CD44, PE-anti-CD69, PE-anti-CD62L and APC-anti-CD4. PE-anti-CCR7 antibodies were purchased from e-Bioscience. A clonotypic mAb specific for the TCR of 3A9 mice, designated “1G12”, a gift from E. Unanue (Washington University, St. Louis, MO), was conjugated with FITC. Lipopolysaccharide (LPS) was from Sigma and Percoll and Ficoll were purchased from GE Healthcare. Endotoxin-free phosphorothioate ODN was synthesized at the Core Facility of the Center for Biologics Evaluation and Research, Food and Drug Administration (Bethesda, MD). The sequence of the CpG ODN used here (#1555) is: (5′-GCTAGACGTTAGCGT-3′).

Tan C., Wandu W.S., Lee R.S., Hinshaw S.H., Klinman D.M., Wawrousek E, & Gery I. (2016). SHEDDING NEW LIGHT ON THE PROCESS OF “LICENSING” FOR PATHOGENICITY BY Th-LYMPHOCYTES. Journal of immunology (Baltimore, Md. : 1950), 198(2), 681-690.

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LRRK2 siRNA Co-Immunoprecipitation

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800 nM of either LRRK2 siRNA (M-006323-02-0010; Dharmacon) or control scrambled siRNA (D-0001206-13-05) was electroporated into HEK-293T cells according to the Lonza Kit V protocol. After 48-h growth at 37°C, cells were harvested and lysed as above for co-IP. Antibody-conjugated Dynabeads (50 μl; 14311D), previously coupled to TRIM1 antibody (PA5-28457; Thermo Fisher Scientific) or IgG isotype control (550326; BD Pharmingen), were incubated with lysates for 16 h at 4°C. Samples were washed, eluted, and immunoblotted as above.

Stormo A.E., Shavarebi F., FitzGibbon M., Earley E.M., Ahrendt H., Lum L.S., Verschueren E., Swaney D.L., Skibinski G., Ravisankar A., van Haren J., Davis E.J., Johnson J.R., Von Dollen J., Balen C., Porath J., Crosio C., Mirescu C., Iaccarino C., Dauer W.T., Nichols R.J., Wittmann T., Cox T.C., Finkbeiner S., Krogan N.J., Oakes S.A, & Hiniker A. (2022). The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity. The Journal of Cell Biology, 221(4), e202010065.

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Anti-CD4 Depletion for Stroke Research

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Mice were injected intraperitoneally (i.p.) with 5mg/kg of an anti-CD4 depletion antibody (clone GK 1.5, eBioscience, San Diego, CA) or IgG isotype control (BD Biosciences PharMingen). The dose was based on prior literature that found that i.p. injection of this antibody (0.2mg/kg/day on 2 consecutive days and 5mg/kg a week apart) leads to depletion of CD4 T lymphocytes in the peripheral blood in mice (Mochimaru et al., 2008 (link)). In order to ensure adequate depletion, we administered 5mg/kg on days 3 and 4 following stroke, at a time of high parenchymal T cell infiltration. CD4 T cell depletion was confirmed by flow cytometry using an anti-CD4 clone (RM4-5) (Supplementary figure 2).

Harris N.M., Roy-o’Reilly M., Ritzel R.M., Holmes A., Sansing L.H., O’Keefe L.M., McCullough L.D, & Chauhan A. (2020). Depletion of CD4 T cells provides therapeutic benefits in aged mice after ischemic stroke. Experimental neurology, 326, 113202.

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Comprehensive Flow Cytometric Analysis of Lymphocytes

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Peripheral blood lymphocytes, lymph node cells, spleen cells, and intrahepatic lymphocytes were analysed using flow cytometry as described [32 (link)]. The following antibodies were used in this study: FITC, R-phycoerythrin, Allophycocyanin-conjugated anti-CD3(555916, BD); FITC, R-phycoerythrin, PE-Cy5-conjugated anti-CD56(340410, BD); FITC, R-phycoerythrin -conjugated anti-CD4(555346, BD) and anti-CD8(557085, BD): FITC-conjugated anti-CD27 (555440, BD), CD28(16-0289-81, ebioscience), CD45RA(555488, BD), CD45RO(561887, BD), CD38(555459, BD), CD69(555530, BD), CD95(555673, BD), CD62L(555543, BD), CD16(555406, BD), CD25(560990, BD), and IgG isotype control (BD PharMingen, San Diego, CA). All data were acquired using a LSRII instrument (BD Biosciences) and were analyzed by using FlowJo (Treestar software). All samples were incubated with an Fcγ blocking antibody (14-9161-73, ebioscience) before staining.

Shi Y., Zhang P., Wang G., Liu X., Sun X., Zhang X., Li H., Qi J., Ding L., Li T., Zhang R., Chen Y., Zhou J., Lv G, & Tu Z. (2018). Description of organ-specific phenotype, and functional characteristics of tissue resident lymphocytes from liver transplantation donor and research on immune tolerance mechanism of liver. Oncotarget, 9(21), 15552-15565.

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Endothelial Cell Heterogeneity in Vasculitis

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The heterogeneity of biologic properties in EC of different origins (e.g. EC derived from large/medium/small vessels or from different organs/tissues) has been well characterized [22 (link)]. Since HSP is small vessels vasculitis that occurs mostly in cutaneous capillaries and postcapillary venules [3 (link),4 (link)], we used primary human dermal microvascular endothelial cells (HMVEC-d, Lonza, Walkersville, MD, USA) as the targets for subsequent experiments. Of note, all studies were performed on cells between passages 3 and 11. HMVEC-d were seeded on gelatin-coated 12-well plates at a density of 5×10⁴ cells/well. When the cellular growth became confluent, plasma of patients with acute HSP, plasma of healthy controls, or complete culture medium (EGM-2MV, Clonetics) alone (100 μl) were added to each well to co-culture with HMVEC-d for further 48 hr. Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Yang Y.H., Tsai I.J., Chang C.J., Chuang Y.H., Hsu H.Y, & Chiang B.L. (2015). The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura. PLoS ONE, 10(3), e0120411.

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Leukocyte Surface Marker Staining

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Cell surface staining procedures were carried out using the BD standard protocol: aliquots of 100 μL fresh blood were added into fluorescence‐activated cell sorting (FACS) tubes with pre‐mixed antibody cocktails. Mouse anti‐human IgG monoclonal antibodies (mAbs) conjugated with fluorophores, which were excited at FITC, PE, PerCP, and APC channels, were used to stain leukocytes (Table S2). An autofluorescence tube containing only whole blood and an IgG isotype control (BD Australia) tube were prepared for each AIBL sample. Optimal concentration for antibodies was determined by titration tests. For intracellular staining, cells were fixed and permeabilized before mixing with antibody cocktail according to the BD standard protocol. Flow cytometry was performed by using FACSCalibur (BD Biosciences). All flow data were stored in digital form and were analyzed using FlowJo software (V10, FlowJo, LLC).

Huang X., Li Y., Fowler C., Doecke J.D., Lim Y.Y., Drysdale C., Zhang V., Park K., Trounson B., Pertile K., Rumble R., Pickering J.W., Rissman R.A., Sarsoza F., Abdel‐Latif S., Lin Y., Doré V., Villemagne V., Rowe C.C., Fripp J., Martins R., Wiley J.S., Maruff P., Mintzer J.E., Masters C.L, & Gu B.J. (2022). Leukocyte surface biomarkers implicate deficits of innate immunity in sporadic Alzheimer's disease. Alzheimer's & Dementia, 19(5), 2084-2094.

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Isolation and Characterization of Porcine Cardiac Adipose-Derived Mesenchymal Stem Cells

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Primary porcine cATMSC were obtained from pigs (Large White × Landrace) undergoing cardiac surgery (n = 39) by cardiac adipose biopsy (1.63 ± 0.69 g), as previously described [7 (link)]. Tissue samples were washed from debris and red blood cells in phosphate-buffered saline (PBS; Oxoid), and digested for 30 min with 0.05% collagenase (Type II; Gibco Invitrogen Corp.) at 37 °C under mild shaking. Digestion was quenched with α-MEM (Sigma Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM l-glutamine, 1% penicillin-streptomycin (P/S) (all from Gibco Invitrogen Corp.) and 5 μg/ml Plasmocin™ (Invivogen) (α-MEM-FBS). Cells were then centrifuged at 1200×g for 10 min and cultured by adherence in α-MEM-FBS at 37 °C 5% CO₂. The purity of established cell cultures was analysed at passages 4–8 by immunostaining with monoclonal antibodies against CD105 (Abcam), CD44, CD45, CD14 (AbD Serotec), CD29, CD34, CD90 and CD106 or IgG isotype control (BD Pharmingen) using a FACSCanto II flow cytometer (BDBiosciencies) and the FACSDiva™ software (BD Biosciencies).

Monguió-Tortajada M., Prat-Vidal C., Moron-Font M., Clos-Sansalvador M., Calle A., Gastelurrutia P., Cserkoova A., Morancho A., Ramírez M.Á., Rosell A., Bayes-Genis A., Gálvez-Montón C., Borràs F.E, & Roura S. (2021). Local administration of porcine immunomodulatory, chemotactic and angiogenic extracellular vesicles using engineered cardiac scaffolds for myocardial infarction. Bioactive Materials, 6(10), 3314-3327.

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Characterization of Cellular Receptors

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HT-3 cervical epithelial cells were obtained from the American Type Tissue Collection (ATCC, Manassas, VA). Jβ2.7 LFA-1+ and LFA-1- Jurkat cells were kindly provided by Catarina Hioe (NYU, New York, NY). Anti-CD18 monoclonal antibody (Mab), H52, was a gift from Dr. James Hildreth (University of California, Davis, Davis, CA). Anti-CD11a Mab (38) was purchased from Abcam (Cambridge, MA). The broadly neutralizing anti-gp120 Mab, b12,^{24 (link)} was kindly provided by Dr. Dennis Burton (The Scripps Institute, La Jolla, CA). IgG Isotype control was purchased from Becton Dickinson (Franklin, Lakes, NJ). FITC conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA). Anti-T7 tail fiber Mab was purchased from Novagen (San Diego, CA). Anti-His Mab was purchased from GE Healthcare Biosciences (Pittsburgh, PA). HRP-conjugated goat anti-mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO).

Guedon J.T., Luo K., Zhang H, & Markham R.B. (2015). Monoclonal And Single Domain Antibodies Targeting β-Integrin Subunits Block Sexual Transmission of HIV-1 in in vitro and in vivo Model Systems. Journal of acquired immune deficiency syndromes (1999), 69(3), 278-285.

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