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Directly conjugated antibodies

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Directly conjugated antibodies are a type of lab equipment used in various biological and biomedical applications. They consist of an antibody molecule directly coupled to a reporter molecule, such as a fluorescent dye or an enzyme. This direct conjugation allows for the easy detection and visualization of target analytes in samples without the need for additional labeling steps.

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Lab products found in correlation

Kaluza 1, by Beckman Coulter (1 mentions) Compbeads set, by BD (1 mentions) Cytoflex, by Beckman Coulter (1 mentions) Facs diva, by Tree Star (1 mentions) Live dead fixable aqua stain, by Thermo Fisher Scientific (1 mentions) Trustain fcx antibody, by BioLegend (1 mentions) Facscanto, by BD (1 mentions) Mitosox red, by Thermo Fisher Scientific (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Rnase a, by Thermo Fisher Scientific (1 mentions) Accutase, by Innovative Cell Technologies (1 mentions) Facsaria, by BD (1 mentions)

3 protocols using directly conjugated antibodies

1

Multimarker Characterization of hOSSCs

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Co-expression of six epitopes, including the CD34, CD73, CD90, CD105, CD146, and CD166 was analyzed on trypsinized hOSSCs and reference cells using a batch of directly conjugated antibodies (all from BD Bioscience, Albertslund, Denmark) using the CytoFLEX and the Kaluza 1.3 software package (both from Beckman Coulter, Copenhagen, Denmark) as previously described.[21 ,22 ] Compensation values were established for each run to control for the bleed-through utilizing the BD CompBeads Set (BD Bioscience) and the AutoComp Wizard in Summit 6.1 (Beckman Coulter), and the Kaluza 1.3 when analyzing the data. The gating protocol included a demarcation of each cell population from the cellular debris followed by the determination of the area of stable flow and selection of single cells. A cut-off value representing the top 2.5 percentile of the fluorescence minus one (FMO) control was then used to establish the positive population for each of the markers.[23 ,24 ]
Liu L., Yu Y., Peng Q., Porsborg S.R., Nielsen F.M., Jørgensen A., Grove A., Bath C., Hjortdal J., Christiansen O.B., Fink T, & Zachar V. (2020). Distribution of Stromal Cell Subsets in Cultures from Distinct Ocular Surface Compartments. Journal of Ophthalmic & Vision Research, 15(4), 493-501.
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2

Flow Cytometric Analysis of Immune Cells

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Cells were washed in PBS and stained with Live/Dead Fixable Aqua stain (Invitrogen) for 30 min in the dark followed by washing with FACS wash buffer (PBS containing 05% FBS, 2 mM EDTA and 0.1% sodium azide). As a positive control for dead cell staining, 5×105 cells were treated with heat at 65°C for 2 min, placed on ice for 2 min and mixed with equal number of live cells. Single-cell suspensions were incubated with TruStain FcX antibody (BioLegend) to block Fc receptors and then stained with directly conjugated antibodies (BD Bioscience; BioLegend). Flow cytometric analysis was performed on a FACSCanto (BD Biosciences) and 100,000 events were recorded for each sample. Data were analyzed using FACS Diva and FlowJo software (Tree Star). Gating was on Aqua Viability Stain negative CD45 positive populations, in which further markers were analyzed.
Wondimu A., Liu Y., Yan S., Bobb D., Ma J.S., Chakrabarti L., Radoja S, & Ladisch S. (2014). Gangliosides drive the tumor infiltration and function of myeloid-derived suppressor cells. Cancer research, 74(19), 5449-5457.
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3

Quantification of Stem Cell Markers

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Cells were dissociated with Accutase (Innovative Cell Technologies) and stained with directly conjugated antibodies (BD Biosciences). The antibody used for flow cytometry was SSEA3 (Sigma). For mtROS assessment, cells were stained with MitoSOX Red (Life Technologies) at a final concentration of 20 μM in cell culture medium. Cell sorting was performed on a FACSAria (BD Biosciences). For cell cycle analysis, cells were collected in PBS and fixed in cold 70% ethanol. Followed by RNase A (Ambion) treatment, cells were stained with propidium iodide (50 μg/ml, Invitrogen) in PBS and subjected to FACS analysis according to standard procedures. For post-sort analysis, data was processed using FlowJo (Tree Star) software.
Vera E., Bosco N, & Studer L. (2016). GENERATING LATE-ONSET HUMAN iPSC-BASED DISEASE MODELS BY INDUCING NEURONAL AGE-RELATED PHENOTYPES THROUGH TELOMERASE MANIPULATION. Cell reports, 17(4), 1184-1192.
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