Dhr 123

Manufactured by MoBiTec

Sourced in Germany

The DHR)-123 is a piece of laboratory equipment. It is designed to perform specific functions within a research or testing environment. The core function of this product is to provide precise measurements and analysis, but the specific intended use is not detailed here.

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Lab products found in correlation

96 well round bottom microtiter plate, by Corning (5 mentions) Accurie c6 flow cytometer, by BD (2 mentions) Cellquest pro software, by BD (1 mentions) Flow cytometry, by BD (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Facscalibur, by BD (1 mentions)

7 protocols using dhr 123

Measuring Macrophage Reactive Oxygen Species

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ROS generation was induced in 96-well round-bottom microtiter plates (Corning, NY, USA). Day 4 macrophages (1 Â 10 5 /well) in RPMI medium were incubated with killed non-opsonized E. coli bacteria. Day 4 macrophages, after washing off the PMN-DGP, were stimulated with bacteria (50 bacteria/cell) for 15 min (37 C, 5% CO 2 ). For the detection of ROS, dihydrorhodamine 123 (DHR 123, Mobitec, Goettingen, Germany) was added to the cells (750 ng/ml final).
After incubation, cells were washed with RPMI medium and suspended in medium containing PI
(2 mg/ml final) to exclude dead cells from the analysis. The relative amount of generated ROS was determined flow cytometrically by the median green fluorescence intensity of gated cells after acquisition of 20 000 viable macrophages.

Hussen J., Koy M., Petzl W, & Schuberth H.J. (2016). Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets. Innate immunity, 22(2).

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Quantifying Leukocyte ROS Generation

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ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) [32 (link)]. Stimulated and non-stimulated leukocytes (1 × 10⁶/well) were incubated for 20 min (37 °C, 5 % CO₂) with the ROS-sensitive dye dihydrorhodamine (DHR)-123, (750 ng/ml final, Mobitec, Goettingen, Germany). After incubation, cells were washed with MIF buffer, and the percentage of ROS-positive cells and the relative amount of generated ROS was determined by flow cytometry (Accurie C6 flow cytometer, BD Biosciences) after acquisition of 100 000 events (Fig. 2A-B).

, & Hussen J. (2021). Bacterial species-specific modulatory effects on phenotype and function of camel blood leukocytes. BMC Veterinary Research, 17, 241.

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Measurement of PMN ROS Production

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Isolated PMNs (∼1 × 10⁵) were incubated with dihydrorhodamine 123 (DHR123) (1 μM) (MoBiTec GmbH, Goettingen, Germany) in 0.5 mL HBSS buffer to detect the production of ROS as previously described (Kaufmann et al., 2012 (link)). After priming of the cells with TNF (10 ng/mL) for 5 min, PMNs were stimulated with fMLP (10^-7 M) for another 5 min. As a positive control, cells were stimulated with PMA. Activation of cells was terminated by putting the tubes in ice water. Production of ROS was determined by flow cytometry (BD, Heidelberg, Germany). DHR fluorescence (530 nm) was measured for gated PMN on a FACScan 9235 (Becton Dickinson Immocytometry Systems, Germany) and analyzed through Cell Quest Pro software (BD Biosciences, United States). Data were collected for 5000 events from each sample and are expressed as mean fluorescence intensity (MFI).

Buchheim J.I., Matzel S., Rykova M., Vassilieva G., Ponomarev S., Nichiporuk I., Hörl M., Moser D., Biere K., Feuerecker M., Schelling G., Thieme D., Kaufmann I., Thiel M, & Choukèr A. (2019). Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight. Frontiers in Physiology, 10, 85.

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Leukocyte ROS Generation Assay

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ROS generation was measured in 96-well round-bottom microtiter plates (Corning, NY, USA) as previously described (37 (link)). Leukocytes separated from heat-stressed whole blood or heat-stressed leukocytes (1 × 10⁶/well in RPMI medium) were incubated in duplicates for 20 min (37°C, 5% CO₂) with heat-killed S. aureus (30 bacteria/cell) in the presence of the ROS-sensitive dye dihydrorhodamine (DHR)-123 (1 μg/mL final, Mobitec, Goettingen, Germany). After incubation, labeled cells were washed with PBS and the relative amount of generated ROS was determined by flow cytometry (Accuri C6 flow cytometer, BD Biosciences) after the acquisition of 100 000 events (gated leukocytes).

, & Hussen J. (2021). Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro. Frontiers in Veterinary Science, 8, 647609.

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Camel Granulocyte ROS Production Assay

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The production of ROS metabolites was measured in 96-well round-bottom microtiter plates (Corning, NY, USA) as previously described [29 (link)]. Camel granulocytes (1 × 10⁶/100 μL/well) were incubated in 50 μL RPMI culture medium alone or in a medium containing T. evansi (4 × 10⁶ parasite/mL) for 30 min (37 °C, 5% CO₂). After 15 min of incubation, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells at a final concentration of 750 ng/mL. The cells were washed in RPMI medium, and ROS production was analyzed by flow cytometry.

Hussen J., AL-Jabr O.A., Alkuwayti M.A., Alrabiah N.A., Falemban B., Alouffi A., Al Salim W.S., Kamyingkird K, & Desquesnes M. (2023). A Flow Cytometry Study of the Binding and Stimulation Potential of Inactivated Trypanosoma evansi toward Dromedary Camel Leukocytes. Pathogens, 13(1), 21.

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Monocyte ROS Determination by Flow Cytometry

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For monocyte identification, separated leukocytes were firstly labeled with anti-CD14 antibody. For this, 100 µL per well of 1 × 10⁶ leukocyte suspension were incubated with an APC-conjugated mouse IgG2a against human CD14. After 15 min at 4 °C, the cells were washed twice with MIF buffer. Labeled leukocytes were then incubated (20 min; 37 °C, 5% CO₂) with 500 ng/mL dihydrorhodamine-123 (DHR-123, Mobitec, Goettingen, Germany). After that, the cells were washed once with PBS (300× g for 3 min) and the median fluorescence intensity of FL1 (indicative for ROS amount) was determined by flow cytometry (Accurie C6 flow cytometer, BD Biosciences, Heidelberg, Germany). The fluorochromes allophycocyanin (APC) and DHR-123 were excited by the red and the blue lasers and detected in FL-1 and FL-4, respectively.

Hussen J, & Al-Sukruwah M.A. (2022). The Impact of the Animal Housing System on Immune Cell Composition and Function in the Blood of Dromedary Camels. Animals : an Open Access Journal from MDPI, 12(3), 317.

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Camel Leukocyte ROS Generation

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ROS generation was performed in 96-well round-bottom microtiter plates (Corning, NY, USA) as described earlier [28 (link)] with modifications. Separated camel leukocytes (1 × 10⁶ / well) in RPMI medium were incubated with heat killed Staphylococcus aureus (50 bacteria/cell) for 20 min (37 °C, 5% CO₂). For the detection of ROS, dihydrorhodamine (DHR) 123 (Mobitec, Goettingen, Germany) was added to the cells (150 ng / ml final). To identify monocyte subsets, cells were labeled with monoclonal antibodies to CD14 and MHCII (see above). After washing, cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, California, USA). The relative amount of generated ROS was determined by the mean green fluorescence intensity of gated monocyte subsets (based on CD14 and MHCII expression) after acquisition of 100,000 events (n = 15 animals).

Hussen J., Shawaf T., Al-Mubarak A.I., Al Humam N.A., Almathen F, & Schuberth H.J. (2020). Dromedary camel CD14high MHCIIhigh monocytes display inflammatory properties and are reduced in newborn camel calves. BMC Veterinary Research, 16, 62.

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