Concentrations of sOPN were measured using a FlowCytomix Simplex Kit (eBioscience, Vienna). The kit consisted of fluorescent microspheres with an emission wavelength of 700 nm. Microspheres were coated with specific antibodies raised against each of the analytes. They also contained a biotin-conjugated second antibody and streptavidin-phycoerythrin emitting at 575 nm. Samples were run on a Cell Lab QuantaTM SC-MPL (Beckman Coulter). Samples were acquired by Cell Lab QuantaTM SC-MPL software (Beckman Coulter) and analysed using FlowCytomixTM Pro 3.0 software (eBioscience). Total protein concentration was determined using the Bradford method.
Cell lab quantatm sc mpl
Manufactured by Beckman Coulter
Sourced in United States
The Cell Lab QuantaTM SC-MPL is a flow cytometry system designed for cellular analysis. It features multi-parameter detection capabilities to quantify various cellular properties, such as size, granularity, and fluorescence signals. The system provides reliable and consistent data for research applications.
Automatically generated - may contain errors
6 protocols using cell lab quantatm sc mpl
1
Quantification of Soluble Osteopontin Levels
2
Quantifying Soluble VCAM-1 Levels
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Concentrations of sVCAM-1 in samples were measured using a FlowCytomix Simplex Kit (eBio-science, Vienna). The kit consists of fluorescent microspheres (diameter: 4 μm, emission wavelength at 700 nm) coated with specific antibodies raised against sVCAM-1. It also contains a biotin-conjugated second antibody and straptavidine-phycoerythrin emitting at 575 nm. Samples were run on a Cell Lab QuantaTM SC-MPL (Beckman Coulter). Samples were acquired by the Cell Lab QuantaTM SC-MPL software (Beckman Coulter) and analysed using FlowcytomixTM Pro 3.0 software (eBioscience). Electronic volume vs. side scatter gating was employed to exclude any sample particles other than 4 μm microspheres. A seven point standard curve ranging from 2.74 to 2000 ng/ml was obtained by serial dilution of the reconstituted lyophilized standard. The lower limit of detection was 0.9 ng/ml.
3
Gamete Size Measurement for Parthenogenetic Strains
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Gamete size was measured for representative strains of each parthenogenetic phenotype found in the segregating population (P+ and P-) (S3 Table ). Synchronous release of gametes was induced by transferring each gametophyte to a humid chamber in the dark for approximately 14 hours at 13°C followed by the addition of fresh culture medium under strong light irradiation. Gametes were concentrated by phototaxis using unidirectional light, and collected in Eppendorf tubes. Gamete size was measured by impedance-based flow cytometry (Cell Lab QuantaTM SC MPL, Beckman Coulter). A Kruskal-Wallis test (α = 5%) followed by a posthoc Dunn’s test for pairwise comparisons were performed using R software to compare female and male gamete size (S16 Table ).
4
Generation of Cassava Autotetraploids
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Cassava cultivar NZ199 autotetraploid genotype was ployploidized artificially by the use of colchicine applied as a solution of 0.001% to lateral buds over a period of 72 h. The emerging shoots were screened for the formation of chimeras or total tetraploids with the chimeras eliminated. To identify autotetraploids, buds were observed for standard chromosome counting and flow cytometric analysis (CellLab QuantaTM SC MPL, Beckman, USA) [24] (link) in addition to observing leaf shape. The shoots were propagated vegetatively and grown at the Cassava Germplasm Pool, Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences. Functional leaves on the top of cassava diploid and autotetraploid plants grown for 180 d at CGB were sampled for protein extraction. One leaf was collected from one cassava plant, and three leaves were collected.
5
Evaluating Apoptosis via FACS Analysis
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Fluorescence-activated cell-sorting (FACS) analysis for apoptosis was done 48 h post-transfection, using an annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system obtained from BD Biosciences (San Diego, CA, USA) and a Cell Lab QuantaTM SC MPL (Beckman Coulter, Fullerton, CA, USA). Cells were stained with annexin V-FITC only (early apoptotic) or both annexin V-FITC and 7-AAD (late apoptotic) and considered to be total apoptotic cell fractions.
6
Apoptosis Assessment by FACS Analysis
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Fluorescence-activated cell-sorting (FACS) analysis for apoptosis was done at 24 and 48 hours post-transfection, using an annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD) staining system (BD Biosciences, San Diego, CA) and a Cell Lab QuantaTM SC MPL (Beckman Coulter, Fullerton, CA). Cells stained with annexin V-FITC only (early apoptotic), or both annexin V-FITC and 7-AAD (late apoptotic) were considered to be apoptotic cell fractions.
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