Expression of red cell surface markers was analyzed on a FACS Calibur instrument (Becton Dickinson, Lincoln Park, NJ, USA). Cells were washed and re-suspended in PBS containing 0.1% BSA (FACS buffer), and stained with fluorescence-labeled antibodies. Data analyses were performed with FlowJ software (TreeStar). Flow cytometry antibodies were purchased from BD Biosciences: APC rat anti-mouse Ter119 (cat# 557909) and PE or FITC labeled rat anti-mouse CD71 (cat# 553267 or 553266, respectively, San Jose, CA, USA). Erythroid subpopulations (nucleated erythroblasts and reticulocytes) were sorted using a FACSAria cytometer (BD Biosciences) based on CD71/Ter119 expressions. CD71+/Ter119+/FSChigh cells were defined as nucleated cells, whereas CD71+/Ter119+/FSClow cells were considered reticulocytes [20 (link)]. To measure hydrogen peroxide (H2O2)-induced red cell lyses, whole blood diluted at 1:500 in PBS was incubated with different concentrations of H2O2 at room temperature for 5 minutes, shook vigorously for 15 seconds, and analyzed immediately on forward scatter channel (FSC) and side scatter channel (SSC) using flow cytometry.
Flow j software
Manufactured by Tree Star
Sourced in United States
Flow J is a software application designed for analyzing and quantifying data from flow cytometry experiments. It provides tools for visualizing and interpreting the results of these analyses.
Automatically generated - may contain errors
4 protocols using flow j software
1
Erythroid Cell Characterization by FACS
2
Annexin V-FITC Apoptosis Assay for Vincristine Sensitivity
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GC SGC7901 cells and variants overexpressing HNF-4α were treated with 0.25 µg/ml vincristine. SGC7901/VCR cells and their variants with knockdown of HNF-4α were treated with 2.5 µg/ml vincristine. Following incubation at 37°C for 24 h with vincristine, the apoptotic cells were analyzed using flow cytometry using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) as described previously (11 (link)). Briefly, cell samples were sequentially incubated with Annexin V-fluorescein isothiocyanate and propidium iodide (PI) following the manufacturer's protocol and then analyzed with a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) using a 530/30 nm signal detector for Annexin V-FITC and a 582/42 nm signal detector for PI. The data were subsequently analyzed by Flow J software (version 7.6.5; Tree Star, Inc., San Carlos, CA, USA). The upper left and lower left quadrants represented late and early apoptosis, respectively. The total apoptosis ratio was calculated by adding the late and early apoptosis proportions.
3
Quantifying Apoptosis with Annexin V-FITC
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An Annexin V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (Thermo Fisher Scientific) was used to evaluate cell apoptosis according to the kit’s instructions. In brief, the cells were incubated with 5 μL propidium iodide and 5 μL Annexin V-FITC at room temperature in the dark for 5 min. Thereafter, the apoptotic rate in cells was examined using a flow cytometer (FACS Verse, BD, Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed by the Flow J software (Tree-star Inc, San Carlos, CA, USA).
4
Intracellular Adriamycin Accumulation Assay
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The intracellular accumulation and retention of Adriamycin was determined using flow cytometry. GC cells and their variants were inoculated into 6-well plates and allowed to adhere overnight at 37°C. Adriamycin (5 mg/ml) was added and cells were incubated at 37°C in Adriamycin-containing RPMI-1640 medium with 10% fetal bovine serum for 1 h. To detect Adriamycin retention, cells were transferred to Adriamycin-free RPMI-1640 medium with 10% fetal bovine serum for another 1 h and then trypsinized, washed, resuspended in phosphate buffered saline (PBS) and subjected to flow cytometry. A flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) was used with a 582/42 nm signal detector for intracellular Adriamycin. The data were subsequently analyzed by Flow J software (version 7.6.5; Tree Star, Inc.). Mean fluorescence intensity of Adriamycin was obtained and expressed as the mean ± standard error of the mean. The Adriamycin-releasing index was calculated as 100% × (mean fluorescence intensity of accumulation-mean fluorescence intensity of retention)/(mean fluorescence intensity of accumulation). Experiments were performed in triplicate.
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