Human primary chondrocytes subjected to different treatments were collected using trypsin without EDTA (Thermo Fisher Scientific) and washed twice with PBS. For the apoptosis assay, the cells were stained using an Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) Apoptosis Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions and then detected using a BD FACS flow cytometer (BD Biosciences). For the cell cycle assay, the cells were fixed overnight in absolute ethanol, washed twice, and incubated with PI/RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA), and the specimens were then detected using a BD FACS flow cytometer (BD Biosciences).
Facs flow cytometer
Manufactured by BD
Sourced in United States, Germany, China
The FACS flow cytometer is a laboratory instrument used for the analysis and sorting of cells or particles suspended in a fluid stream. It utilizes the principles of light scattering, light excitation, and electronic detection to characterize and differentiate individual cells or particles as they flow in a single file through a laser beam.
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Lab products found in correlation
Fitc annexin 5 apoptosis detection kit, by BD (13 mentions)
Annexin 5 fitc pi staining kit, by BD (12 mentions)
Propidium iodide, by Merck Group (11 mentions)
Cellquest software, by BD (10 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (5 mentions)
Cellquest pro software, by BD (5 mentions)
Rnase a, by Keygen Biotech (4 mentions)
Cellquest pro, by BD (4 mentions)
Rnase a, by Merck Group (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Rnase a, by Thermo Fisher Scientific (4 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (4 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (4 mentions)
Annexin 5 fitc pi, by BD (4 mentions)
Pi rnase staining buffer, by BD (3 mentions)
Propidium iodide, by BD (3 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Liberase tl, by Roche (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
328 protocols using facs flow cytometer
1
Chondrocyte Apoptosis and Cell Cycle Assays
2
Cell Cycle and Apoptosis Analysis of Kasumi-1 Cells
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1 million Kasumi-1 cells were treated with the IC50 value of the MLEE or MO-AgNPs for 24 hours for the cell cycle analysis. After treatment, the cells were fixed in 70% ethanol for 2 hours. The fixed cells were resuspended in PBS containing 0.1% triton-X and RNase A. The cells were then incubated with propidium iodide (PI) (Sigma) and analysed with the BD-FACS flow cytometer. Further analysis was done using the ModFit program. For the apoptosis assay, 500,000 Kasumi-1 cells were treated with the IC50 of the MLEE or MO-AgNPs for 72 hours. The cells were harvested and further methods were done using the annexin V – FITC apoptosis kit (Sigma Aldrich) according to its manufacturer’s protocol. The cells were then analysed with the BD-FACS flow cytometer.
3
Cell cycle and apoptosis analysis
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Following incubation with wogonin, the cells were washed with phosphate-buffered saline (PBS) and suspended in a propidium iodide (PI) buffer (10 µg/ml PI, 0.5% Tween-20, 0.1% RNase in PBS). The cell cycle was analyzed using a FACS flow cytometer (Becton-Dickinson, San Jose, CA, USA).
For the detection of apoptosis, the Annexin V/PI apoptosis kit (Becton-Dickinson) was used. The cells were washed twice with PBS and suspended with binding buffer. Subsequently, 5 µl of Annexin V-FITC and 10 µl of PI were added followed by the incubation of the cells in the dark. The apoptotic rate was examined using a FACS flow cytometer (Becton-Dickinson).
For the detection of apoptosis, the Annexin V/PI apoptosis kit (Becton-Dickinson) was used. The cells were washed twice with PBS and suspended with binding buffer. Subsequently, 5 µl of Annexin V-FITC and 10 µl of PI were added followed by the incubation of the cells in the dark. The apoptotic rate was examined using a FACS flow cytometer (Becton-Dickinson).
4
Bacterial Viability Assay at Low pH
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Bacteria were grown to stationary phase at 37 °C in LB medium adjusted to pH 5.0. A 1-ml aliquot of culture was then centrifuged for 5 min at 2000 × g, and the resulting pellet resuspended in 1 ml of LB medium adjusted to either pH 2.5 or pH 5.027 (link)28 (link). The suspension was then incubated for 30 min at 37 °C with shaking at 220 rpm. Bacteria were collected by centrifugation as described above, and washed three times with PBS. Cells were then stained for 15 min using a BD cell viability kit, and measured using a BD FACS flow cytometer. The data were analyzed using CellQuest software.
5
Annexin V-FITC/PI Apoptosis Assay
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Cell apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) apoptosis kit (Invitrogen). Briefly, chondrocytes were cultured for 48 h, digested using trypsin without EDTA (Thermo Fisher Scientific), and washed with PBS. The cells were collected and adjusted to a cell density of 1 × 106 cells/ml. Then, cells were stained with Annexin V-FITC and PI for 15 min. At last, a BD FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect the fluorescence intensity.
6
HER2 Expression Analysis in BT-474 Cells
7
Evaluating Cell Invasion and Cell Cycle
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Transwell® chambers were coated with Matrigel™ (BD biosciences) for 30 min at 37 °C. Cells treated with mitomycin were seeded into the upper chambers and 500 μL complete medium was added to the lower chambers. After 48 h, the cells on the lower chambers were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet solution. Images were captured under a microscope. Each group was analyzed in triplicate.
After isolation by trypsinization, cells were rinsed twice using precooled PBS and then fixed in 70% ethanol at −20°C overnight. Subsequently, fixed cells suspended in 50 μg/mL of propidium iodide (KeyGen BioTECH) and 100 μg/mL of RNaseA (KeyGen BioTECH), cells were incubated for 40 min at room temperature and in the absence of light. Flow cytometry was used for determining the cycle of filtered cells.
In the apoptosis assay, cells were washed using PBS and stained with Annexin V-FITC Apoptosis Detection Kit (Affymetrix eBioscience) according to manufacturer instructions. Flow cytometer (BD Biosciences, BD FACS™ Flow Cytometer) was used for cell analysis.
After isolation by trypsinization, cells were rinsed twice using precooled PBS and then fixed in 70% ethanol at −20°C overnight. Subsequently, fixed cells suspended in 50 μg/mL of propidium iodide (KeyGen BioTECH) and 100 μg/mL of RNaseA (KeyGen BioTECH), cells were incubated for 40 min at room temperature and in the absence of light. Flow cytometry was used for determining the cycle of filtered cells.
In the apoptosis assay, cells were washed using PBS and stained with Annexin V-FITC Apoptosis Detection Kit (Affymetrix eBioscience) according to manufacturer instructions. Flow cytometer (BD Biosciences, BD FACS™ Flow Cytometer) was used for cell analysis.
8
Quantifying Early and Late Apoptosis
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The transfected cells in early and late apoptosis stages were quantified with double stained FITC-Annexin V and PI, using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Briefly, cells were collected and resuspended with 500 μl binding buffer, following by incubated with Annexin V and PI in dark situation for 15 min. Then the stained cells were analyzed using BD FACS flow cytometer (BD Biosciences). The percentage of apoptotic cells were calculated and compared between different groups.
9
Apoptosis and Necrosis Assay
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To examine the morphology of apoptotic and necrotic cells, the cells were seeded in 6-well plates at ~70% confluency and subjected to the indicated treatments. For flow cytometry analyses, cells were treated with various chemotherapy drugs as indicated. Cells were harvested, washed with PBS three times, and stained using Annexin V-FITC/PI Apoptosis Assay Kit (BD Pharmingen, San Jose, CA, USA) and DCFH-DA Reactive Oxygen Species Assay Kit (Meilunbio, China) according to the manufacturer’s instructions. Stained cells were analyzed with a BD FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ), and the data were processed using FlowJo software.
10
Cell Cycle Analysis by Flow Cytometry
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Cells were centrifuged at 1,500 rpm and washed twice with ice-cold PBS. Cells were fixed with 70% ethanol and incubated at 4˚C for 30 min. Following fixation, cells were incubated with RNase A (20 µg/ml) for 30 min at 37˚C, and stained with propidium iodide (50 µg/ml; BD Biosciences) for 30 min at room temperature in the dark. Cell cycle analysis was conducted using a BD FACS flow cytometer (BD Biosciences) (14 (link)).
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