Cell lab quanta sc mpl

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.

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 Quanta^TM 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.

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 Quanta^TM 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.

Chemostat cultures were assayed for viability using the FungaLight AM-CFDA (acetoxymethyl ester 5-carboxyfluorescein diacetate)/propidum iodide yeast viability kit (Invitrogen, Carlsbrad, CA) by counting 10,000 cells on a Cell Lab Quanta SC MPL flow cytometer (Beckman Coulter, Woerden, Netherlands) as described previously [51 (link)]. AM-CFDA is a cell-permeant substrate for an intracellular non-specific esterase activity. Hydrolytic cleavage of the lipophilic blocking and diacetate groups of AM-CFDA results in a green fluorescence in metabolically active cells. Propidium Iodide intercalates with DNA in cells with a compromised cell membrane, which results in red fluorescence.

The cell cycle distribution was analyzed using a flow cytometer (Cell Lab Quanta™ SC MPL; Beckman Coulter Immunotech). Briefly, after irradiation and incubation for the indicated period under cytokine-free conditions, the harvested CD34⁺ cells were treated with PBS containing 0.1% Triton X-100 (Wako, Osaka, Japan) and stained with propidium iodide (50 µg/ml; Sigma–Aldrich, St Louis, Missouri, USA), and their DNA contents were measured.

Cells were seeded at a density of about 2 × 10⁵ per well in a 24-well plate. Following the protein transduction, cells were washed with PBS and harvested by adding 250 μl/well of 2.9 mM ethylenediaminetetraacetic acid (EDTA, pH 6.14)^{22 (link)}. Samples were analyzed using a Cell Lab Quanta SC MPL flow cytometer (Beckman Coulter, Fullerton, CA, USA) possessed with 488 nm laser at a speed of 20 μl/min. Digital measurements were analyzed using Cell Lab Quanta SC MPL software version 1.0 (Beckman Coulter). Results are shown as the percentage of either the total cell population displaying green fluorescence or internalization efficiency.

Flow cytometric analysis was performed to measure the quantity of fluorescent microbeads introduced into the cells by the cell−GUV electrofusion method. After electrofusion, HeLa cells were seeded in 35-mm dishes and cultured for two days. The cells were removed by adding 0.25% trypsin–EDTA solution, and then counted using flow cytometer (Cell Lab Quanta SC MPL, Beckman Coulter, USA) equipped with a blue laser (488 nm). A screening gate was set on the electronic cell volume vs. SSC plot, to allow analysis only of cells of a size with a diameter in the range of 11–16 µm.