Cell proliferation was measured with the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the protocol recommended by the manufacturer. For the cellular apoptosis assay, cells were stained using Annexin-V/Dead Cell Apoptosis Kit (Invitrogen) and analyzed on a BD FACSCalibur flow cytometry (BD Biosciences, Franklin Lakes, NJ). For cell cycle analysis, unsynchronized cells were harvested by trypsinization and fixed with 70% ethanol. Cells were then stained with propidium iodide for total DNA content and the cell cycle distribution was then analyzed using a BD FACSCalibur flow cytometry (Becton Dickinson). For cell migration assay, monolayers of cells were grown on 6 well plates before a cell-free region was created using a 10 μl pipette tip. Scratch wound width was measured using a graticule at 0 h and 12 h post treatment. Representative images were taken at these time points using a Carl Zeiss Microscopy at ×5 magnification.
Facscalibur flow cytometry
Manufactured by BD
Sourced in United States, China, Switzerland
The FACSCalibur flow cytometry system is a versatile instrument used for the analysis and sorting of cells and particles. It utilizes laser technology to detect and measure various physical and fluorescent characteristics of samples, enabling researchers to gather data about cell populations and individual cells.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (38 mentions)
Cellquest pro software, by BD (13 mentions)
Propidium iodide, by Merck Group (9 mentions)
Rnase a, by Merck Group (7 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (6 mentions)
Cell cycle detection kit, by Keygen Biotech (6 mentions)
Propidium iodide, by BD (6 mentions)
Microplate reader, by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Annexin 5 fitc, by BD (5 mentions)
Annexin 5 fitc pi apoptosis detection kit, by BD (5 mentions)
Annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (5 mentions)
Hu6 mcs pgk egfp lentiviral vector, by GenePharma (4 mentions)
Cck 8 kit, by Dojindo Laboratories (4 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Fluorescence activated cell sorting (facs), by BD (3 mentions)
Propidium iodide, by Keygen Biotech (3 mentions)
Facscalibur, by BD (3 mentions)
Dcfh da, by Merck Group (3 mentions)
462 protocols using facscalibur flow cytometry
1
Multimodal Cellular Characterization
2
Cell Proliferation, Apoptosis, and Migration Assays
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Cell proliferation was measured with the CCK‐8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the protocol recommended by the manufacturer. For cell cycle analysis, unsynchronized cells were harvested by trypsinization and fixed with 70% ethanol. Cells were then stained with propidium iodide for total DNA content and the cell cycle distribution was then analyzed using a BD FACSCalibur flow cytometry (Becton Dickinson). For the cellular apoptosis assay, cells were stained using Annexin‐V/Dead Cell Apoptosis Kit (Invitrogen) as per the manufacturer's recommendations and analyzed on a BD FACSCalibur flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). For cell migration assay, monolayers of cells were grown on 6 well plates before a cell‐free region was created using a 10 μL pipette tip. Scratch wound width was measured using a graticule at 0 and 12 hours post treatment. Representative images were taken at these time points using a Carl Zeiss Microscopy at ×5 magnification.
3
Cell Apoptosis and Cell Cycle Arrest Analysis
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Cancer cell apoptosis and cell cycle arrest were analyzed using the cell flow cytometry assays. To detect cell apoptosis, 5637 and 253 J cells were seeded into 6-well plates at 30 × 104 cells per well for 24 h. The cells were then treated at different concentrations (0, 50, and 100 μM) for 24 h. Chilled PBS was used to wash the 6-well plates, prior to suspension with binding buffer. The Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) and FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) were used to detect and calculate the apoptosis rate of the cancer cells. For detecting cell cycle arrest, 5637 and 253 J cells were seeded into 6-well plates and treated at two different concentrations (0, 50, and 100 μM) for 24 h. Cells were washed with chilled PBS and collected using a centrifugal method and subsequently fixed with 70% ethanol at 4 °C overnight. Cell samples were then incubated using propidium iodide (PI) and RNase. FACSCalibur flow cytometry (BD Biosciences, San Jose, CA, USA) was then used to identify cell cycle arrest and calculate the number of cells present in the different cell cycle stages. The experiment was performed in triplicate [28 (link)].
4
Apoptosis and Cell Cycle Evaluation
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Cells apoptotic rate was detected by Flow cytometry using Annexin V-FITC Apoptosis Detection Kit (Key-GEN, Nanjing, China) according to the manufacturer’s instructions. Two mL suspension of 105 cells was stained with (Annexin-V-FITC and PI) kit solution in dark for 15 min. The assay then performed using FACSCalibur Flow Cytometry (BD, USA) at 488 nm.
The rate of cells cycle arrest was also detected by Flow cytometry using Cell Cycle Detection Kit (Key-GEN, Nanjing, China). Following its manufacturer’s instructions, 2 mL suspension of 106 cells was fixed with 70% ethyl alcohol, and washed by PBS before staining. The assay performed in FACSCalibur Flow Cytometry (BD, USA) at 488 nm.
The rate of cells cycle arrest was also detected by Flow cytometry using Cell Cycle Detection Kit (Key-GEN, Nanjing, China). Following its manufacturer’s instructions, 2 mL suspension of 106 cells was fixed with 70% ethyl alcohol, and washed by PBS before staining. The assay performed in FACSCalibur Flow Cytometry (BD, USA) at 488 nm.
5
Quantifying Apoptosis via Flow Cytometry
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At 24 h after transfection with miRNA- 203a-3p mimics or negative controls, the cells were washed with PBS and fixed with 70% ethanol for >12 h at 4°C. Propidium iodide (PI) staining solution (500 µl) was then added to the centrifuged cells (845 × g, 3 min) at room temperature, followed by incubation for 30 min in the dark at room temperature. Cell apoptosis was analyzed by FACSCalibur flow cytometry (BD Biosciences). The percent of apoptotic cells was obtained from FACSCalibur flow cytometry (BD Biosciences) which was used for further calculation. The Annexin V/PI Apoptosis Detection Kit (Beijing Solarbio Science & Technology, Co., Ltd.) was used to assess apoptosis.
6
Cell Cycle and Apoptosis Analysis
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Cell cycle and apoptosis analysis had been performed by flow cytometry (FCM). All cells were harvested with 0.25% trypsin/EDTA (Sigma-Aldrich, Saint Louis, MO, USA) after 24 h treatment and washed with cold PBS. To detect the cell apoptosis, cells were stained with propidium iodide (PI, Sigma-Aldrich, Saint Louis, MO, USA) and annexin V (Sigma-Aldrich, Saint Louis, MO, USA) for 15 min kept in dark place and were analysed by FACS Calibur Flow Cytometry (BD Biosciences, NJ, USA). To detect the cell cycles, cells were fixed with cold 75% ethanol (Sigma-Aldrich, Saint Louis, MO, USA) and stained with PI before quantified by FACS Calibur Flow Cytometry (BD Biosciences, NJ, USA). All tests were measured in triplicate.
7
Analyzing Cell Cycle and Apoptosis
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Exponentially growing HepG2 cells were treated with Tan IIA, Nec-1 and z-VAD-fmk mixture at the desired concentrations, following removal of non-adherent cells by gentle washing. After exposure to the drugs for 12 h, the cells were collected and centrifuged at 500×g in a 15 ml tube for 5 min. The cells were then washed with ice-cold PBS and fixed with 70% ethanol for 2 h at −20 °C. The cells were subsequently washed with PBS and treated with 200 μg/ml RNase and 500 μl of PI (20 μg/ml in stock) for 30 min in darkness at room temperature. PI-stained cells were assayed using BD FACSCalibur Flow Cytometry (Palo Alto, CA, USA) and cell cycle distributions (G0-G1, S, and G2-M) were analyzed with BD CellQuest Pro Software (built-in software; San Jose, CA, USA). Apoptosis was detected by staining the cells with an Annexin V-FITC Apoptosis Detection Kit from Strong Biotech Co. (Taipei, Taiwan) following the manufacturer's instructions. Five million HepG2 cells were stained for 15 min with Annexin V- FITC and PI at room temperature in darkness. After staining, the apoptotic cells were counted using BD FACSCalibur Flow Cytometry and the data were processed with the BD CellQuest Pro Software as described above.
8
Enumeration of Lymphocyte Subsets
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T-Lymphocyte subsets in whole blood samples were enumerated using fluoro isothiocyanate (FITC) conjugated CD4 (Becton Dickinson, Bioscience, USA), phycoerythrin (PE) conjugated CD8 (Becton Dickinson, Bioscience, USA) and peridinium-chlorophyll-protein (Per-CP) conjugated CD3 (Becton Dickinson, Bioscience, USA). B-Lymphocyte subsets in whole blood samples were enumerated using phycoerythrin (PE) conjugated CD19 (Becton Dickinson, Bioscience, USA). Flow cytometric analysis was done by FACS Calibur flow cytometry with Cell Quest software (Becton Dickinson Biosciences, USA). An isotype-matched negative control was used with each sample. Forward and side scatter histogram was used to define the lymphocyte population (R1). The absolute counts and percentages of CD3, 4, 8 and 19 subsets of lymphocytes were calculated by multiplying the percentage (results of flow cytometer) by absolute lymphocyte counts (results of CBC).
9
Tracking miR-762 Regulation of Cell Cycle
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ST cells were seeded on a 16-well E-Plate with 5000 cells per well and allowed to grow for 12–24 h. The cells were transfected with miR-762 mimic or miR-762 inhibitor when the cell index reached 1.0–2.0 with three wells per treatment. Cell growth and proliferation were monitored by an xCELLigence RTCA DP instrument (Roche Applied Science, Penzberg, Upper Bavaria, Germany).
The cell cycle was analysed with the Cell Cycle Detection Kit (KeyGEN BioTECH, Nanjing, China). Briefly, 48 h after transfection, the ST cells were fixed in 70% (v/v) ethanol overnight at −20 °C. Following incubation in 50 mg/ml propidium iodide (PI) for 30 min at 4 °C, the cells were analysed using FACSCalibur Flow Cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) and the ModFit software (Verity Software House). The proliferative index was derived by estimating the proportion of mitotic cells from a total of 20,000 cells.
The cell cycle was analysed with the Cell Cycle Detection Kit (KeyGEN BioTECH, Nanjing, China). Briefly, 48 h after transfection, the ST cells were fixed in 70% (v/v) ethanol overnight at −20 °C. Following incubation in 50 mg/ml propidium iodide (PI) for 30 min at 4 °C, the cells were analysed using FACSCalibur Flow Cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) and the ModFit software (Verity Software House). The proliferative index was derived by estimating the proportion of mitotic cells from a total of 20,000 cells.
10
Cellular Uptake and Cytotoxicity of BORT Liposomes
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Cellular uptake of R18-labeled Tf-L-BORT or L-BORT was evaluated in K562 cells. About 4×105 cells were incubated with liposomes at 37°C. After 1-hour of incubation, the cells were then washed three times with PBS, photographed on a Nikon fluorescence microscope (Nikon, Küsnacht, Switzerland) and measured by a FACS Calibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ). To evaluate BORT toxicity, K562 cells were treated by different concentration of free- or L-BORT. Regarding to the chemosensitivity to doxorubicin, the K562/ DOX cells were pre-treated with doxorubicin for 24 hours and then subtoxic BORT formulations were added for additional 24 hours. Cellular viability was assessed by MTT proliferation assays (Promega, Madison, WI).
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