Appropriate isotype control

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Appropriate isotype controls are laboratory reagents used in flow cytometry and other immunological assays to establish baseline signal levels for specific antibody-antigen interactions. They serve as a reference point to differentiate specific binding from non-specific background signals.

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Lab products found in correlation

Facsaria iiu, by BD (1 mentions) Mouse treg staining kit, by Thermo Fisher Scientific (1 mentions) Cd34 pe, by Thermo Fisher Scientific (1 mentions) Facsdiva, by BD (1 mentions) Fixation buffer, by BD (1 mentions) Cd38 alexa fluor 700, by BioLegend (1 mentions) Cd45ra brilliant violet 570, by BioLegend (1 mentions) Cd49f bv421, by BioLegend (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Anti cd63 pe, by BioLegend (1 mentions) Facscalibur, by BD (1 mentions) Data analysis software, by FlowJo (1 mentions) Cell staining buffer, by BD (1 mentions)

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Isotype control (56 citations) Fluorochrome conjugated antibodies and appropriate isotype controls (2 citations)

5 protocols using appropriate isotype control

Regulatory T Cell Profiling in Murine Tissues

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Single cell suspensions from skin, tumor, draining lymph nodes (dLN) and spleen were stained with fluorochrome-conjugated antibodies against CD11b (PA5-79532, ThermoFisher), CD4 (116005, BioLegend), CD8 (100726, BioLegend), CD25 (102011, BioLegend), Foxp3 (320105, BioLegend), Ly6C (128021, BioLegend) or Ly6G (127607, BioLegend) and appropriate isotype controls (BioLegend) for 30 min on ice. Cells were washed and fixed with 1% paraformaldehyde and analyzed on a FACSAriaIIu (BD Biosciences). The frequency of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) was determined using the mouse Treg staining kit (eBioscience).

Pinto N.A., Abba M.C., Laporte L., Pérez Sáez J.M., Blidner A.G., Torres N.I., Morales R.M., Gatto S.G., Bach C.A., Veigas F., García Rivello H.J., Song P., Frederiksen J.H., Rasmussen L.J., Poirier F., Croci D.O., Sundblad V., Rabinovich G.A, & Cerliani J.P. (2023). Galectin-7 reprograms skin carcinogenesis by fostering innate immune evasive programs. Cell Death and Differentiation, 30(4), 906-921.

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Identification of Hematopoietic Cell Markers

Cited 2 times

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Cell surface markers were identified with the following fluorochromes: human hematopoietic lineage markers (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) conjugated to APC (eBioscience), CD34 PE, CD38 Alexa Fluor 700, CD49f BV421, and CD45RA Brilliant Violet 570 (all from BioLegend). Appropriate isotype controls (BioLegend) were used for gating HSCs as described [29 (link), 30 (link)]. In some cases, following surface marker staining, cells were fixed with fixation buffer (BD Biosciences), permeabilized with Transcription Factor Fixation/Permeabilization Buffer kit (eBioscience) and stained with goat anti-human ARID3a, followed by rabbit anti-goat FITC secondary antibody (Invitrogen) to identify ARID3a-expressing cells and/or with H2A.X phopspho (Ser139) APC-Fire750 (Biologend). Human anti-ARID3a peptide specific antibodies were generated and isolated over a peptide-specific column as described [55 (link)]. Cells were pretreated with anti-human CD32 antibody to block Fc receptor binding prior to surface staining to exclude non-specific binding. Doublet exclusion was used to ensure analyses of single cells prior to forward/side scatter gating. Data were collected using an LSRII (BD Biogenics) and FACSDiva (BD Biosciences) software version 4.1 or Stratedigm S1200Ex and CellCapTure acquisition software and were analyzed using FlowJo (Tree Star) software version 10.

Ratliff M.L., Garton J., James J.A, & Webb C.F. (2020). ARID3a expression in human hematopoietic stem cells is associated with distinct gene patterns in aged individuals. Immunity & Ageing : I & A, 17, 24.

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Multiparametric Flow Cytometry Analysis

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The following fluorescently labeled antibodies and appropriate isotype controls (all from BioLegend) were used in this study CD8α (53-6.7), CD4 (GK1.5), IFNγ (XMG1.2), TNF (MP6-XT22), and CD44 (IM7). Data were collected on a BD Symphony in the OHSU Flow cytometry core facility. Data were analyzed using FlowJo software (version 9.9.6).

Harbour J.C., Lyski Z.L., Schell J.B., Thomas A., Messer W.B., Slifka M.K, & Nolz J.C. (2021). Cellular and humoral immune responses in mice immunized with Vaccinia virus expressing the SARS-CoV-2 spike protein. Journal of immunology (Baltimore, Md. : 1950), 206(11), 2596-2604.

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Exosome Conjugation and Flow Cytometry

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Flow cytometry was done as described earlier by (Bhatnagar et al. 2007 (link))). Briefly, 5 μg CUR-EXO were incubated with 4 μm-diameter latex beads in PBS (50μl) at RT for 20 min. The volume was increased to 300 μl and the beads were incubated at 4°C overnight under gentle agitation. The reaction was stopped by incubation in 100 mM glycine (30 min). Coated beads were washed thrice and labelled with anti-CD63 PE and appropriate isotype control (Biolegend). After that, the beads were washed thrice and analyzed by flow cytometry (BD Accuri™ C6 Flow Cytometer, New Jersey, USA).

Kalani A., Kamat P., Chaturvedi P., Tyagi S, & Tyagi N. (2014). Curcumin-Primed Exosomes Mitigate Endothelial Cell Dysfunction during Hyperhomocysteinemia. Life sciences, 107(0), 1-7.

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Immune Cell Phenotyping by Flow Cytometry

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IMG cells were grown on a 10-cm tissue culture dish until 80 % confluent. After addition of 5 mL fresh media, cells were lifted off the dish using a cell scraper. IMG cells were resuspended to 1 × 10⁸ cells/mL and incubated with fluorescently conjugated CD11b (Alexa 647 conjugate; Serotec), F4/80 (allophycocyanin (APC) conjugate; Caltag), or appropriate isotype control (BioLegend) antibodies (1:10 dilution) in the dark for 15 min at 4 °C. Cells were then washed three times with 2 mL cell-staining buffer (BioLegend). After washing, cells were resuspended in cell-staining buffer and were analyzed by flow cytometry (FACSCalibur, BD Biosciences). Acquired data were analyzed using FlowJo data analysis software (FlowJo, LLC).

McCarthy R.C., Lu D.Y., Alkhateeb A., Gardeck A.M., Lee C.H, & Wessling-Resnick M. (2016). Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta. Journal of Neuroinflammation, 13, 21.

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