MDCK and MDCK-pAbcb1 cells were seeded in six-well plates, and cells were washed with PBS and rhodamine 123 (Rho123; 5 μM) (Sigma-Aldrich, Castle Hill, Australia) was added in the presence and absence of verapamil (100 μM) to identify P-gp mediated Rho123 accumulation. To study the time-course of Rho123 accumulation, cells were harvested at 15, 30, 60 and 120 min after the addition of Rho123 and accumulation was measured using a FACS Calibur (BD Biosciences, Bedford, MA) with CellQuest Prosoftware. Data were collected for a minimum of 10,000 gated events per sample as geometric mean fluorescent intensity for all samples.
Rho123
Manufactured by BD
Sourced in China
Rho123 is a fluorescent dye used for cell labeling and analysis. It is a cationic dye that accumulates in the mitochondria of living cells, allowing for the assessment of mitochondrial membrane potential.
Automatically generated - may contain errors
Lab products found in correlation
Rho123, by Merck Group (3 mentions)
Rho123, by Beyotime (1 mentions)
Flow cytometry, by BD (1 mentions)
Cellquest software version 5, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Rhodamine 123 rho 123, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Facsverse, by BD (1 mentions)
4 protocols using rho123
1
P-gp Mediated Rhodamine 123 Accumulation
2
Evaluating P-gp Efflux and Apoptosis
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P-gp efflux activity was determined by intracellular accumulation of rhodamine 123 (Rho123, Beyotime, China), a substrate of P-gp. After 48 h of transfection, cells were incubated with 4 µg/ml Rho123 for 1 h or 10 min at 37°C in the dark, then washed twice with PBS and subsequently analyzed using a flow cytometry (BD).
Cell apoptotic analysis were assessed by double-staining with Annexin V-FITC and propidium iodide (PI, Vazyme, Nanjing, China). Briefly, cells were harvested after 48 h of ADM treatment, washed with PBS, resuspended in 500 µl of binding buffer, added with 5 µl of Annexin V-FITC and 5 µl of propidium iodide, incubated for 10 min in the dark, and analyzed using a flow cytometer. The Annexin V-positive cells were considered apoptotic cells and analyzed using the FlowJo software. All of assays were carried out in triplicate in three independent experiments.
Cell apoptotic analysis were assessed by double-staining with Annexin V-FITC and propidium iodide (PI, Vazyme, Nanjing, China). Briefly, cells were harvested after 48 h of ADM treatment, washed with PBS, resuspended in 500 µl of binding buffer, added with 5 µl of Annexin V-FITC and 5 µl of propidium iodide, incubated for 10 min in the dark, and analyzed using a flow cytometer. The Annexin V-positive cells were considered apoptotic cells and analyzed using the FlowJo software. All of assays were carried out in triplicate in three independent experiments.
3
Rho 123 Accumulation Analysis
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The Rho 123 accumulation was determined by flow cytometry analysis as previously described (36 (link)). Following the treatment above, the cells in 24-well plates (2×105 cells/well) were incubated with 50 mM Rho 123 (Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. The reaction was terminated at 5 min of incubation on ice. Subsequently, the fluorescent of Rho 123 was detected by FACS Calibur flow cytometer (BD Biosciences). Cell Quest software version 5.1 (BD Biosciences) was used to perform the analysis.
4
Flow Cytometry Analysis of Apoptosis and Mitochondrial Potential
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Flow cytometry (FCM) was used to analyze cell apoptosis and mitochondrial membrane potential (ΔΨm) by staining the cells with propidium iodide (PI) and rhodamine-123 (Rho 123) (both from Sigma-Aldrich; Merck KGaA), respectively. In total, 1×105 SPC-A1 cells were cultured in 6-well plates for 24 h. Following treatment with #2714 for 48 h, SPC-A1 cells were incubated with 50 µl/ml PI solution, 0.1% Triton X-100 and 0.1% sodium citrate, or 5 µg/ml Rho 123 at 37°C with 5% CO2 for 30 min in darkness. PI-stained and Rho 123-stained cells were analyzed using a flow cytometer (FACS Verse; BD Biosciences). Data were analyzed using FlowJo software Version 7.6 (FlowJo LLC, Ashland, OR, USA).
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