Prior to seeding, baseline confirmation of BM-MSC phenotype was confirmed via fluorescence-activated cell sorting (FACS) analysis. For the present study, SSEA-4, CD73, CD90, CD105, and CD146, known markers of stem cells [27 (link), 28 (link)], were analyzed to determine the immunophenotypic profile of the cells before and after culture on the various substrates. Single-cell suspensions (1–2 × 106) were incubated for 30 minutes at 4 °C with 100-μl aliquots (1:10 dilution) of each primary mouse antibody (SSEA-4, CD73, CD90, CD105, and CD146; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Non-specific isotype IgG was used as a negative control. Antibody-labelled cells were washed twice with staining buffer (PBS containing 5 % fetal calf serum and 0.01 % sodium azide) and then incubated for 20 minutes at 4 °C with 20 μg/ml secondary antibody (FITC-conjugated goat anti-mouse IgG; Santa Cruz Biotechnology, Inc.). Cells were then washed twice with staining buffer and either analyzed immediately or fixed by using 400 μl/tube of 1 % paraformaldehyde in PBS and stored at 4 °C until analyzed. At least 10,000 events per sample were acquired by using a Becton Dickinson FACStarplus flow cytometer to determine the percentage of positively stained cells.
Facstar plus flow cytometer
Manufactured by BD
Sourced in United States
The FACStar Plus flow cytometer is an analytical instrument designed to quantify and characterize cells and particles in a fluid stream. It utilizes laser technology to detect and measure various physical and fluorescent properties of individual cells as they pass through the instrument's flow chamber. The FACStar Plus provides data on cell size, granularity, and the presence of specific cell surface markers or intracellular molecules, enabling detailed analysis of complex cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (4 mentions)
Facsdiva software, by BD (4 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Facscan flow cytometer, by BD (2 mentions)
Fluorescence conjugated antibodies, by BD (2 mentions)
Pe conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Accutase, by STEMCELL (2 mentions)
Pe conjugated ssea1, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc pi, by BD (2 mentions)
Ssea4, by Santa Cruz Biotechnology (1 mentions)
Cd105, by Santa Cruz Biotechnology (1 mentions)
Cd146, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Dcfh da probe, by Nanjing Jiancheng (1 mentions)
Jc 1 dye, by Beyotime (1 mentions)
Fluorescence microplate reader, by Molecular Devices (1 mentions)
H2dcfda, by Merck Group (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
57 protocols using facstar plus flow cytometer
1
Confirming BM-MSC Phenotype via FACS
2
Intracellular ROS and Apoptosis Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intracellular reactive oxygen species (ROS) was assessed with the DCFH-DA probe (Nanjing Jiancheng, China). Briefly, 10 μM DCFH-DA was incubated with cell suspensions for 30 min and then residual probe was removed by washing with PBS. The cellular fluorescence intensities were measured on a fluorescence microplate reader (Molecular Devices, USA) with excitation and emission wavelengths set at 488 nm and 525 nm, respectively. Cell apoptosis was analyzed through annexin V-PI staining and JC-1 assay. After treatment, cell precipitate was collected through trypsinization and centrifugation. JC-1 dye (Beyotime, China) diluted with culture medium were added to the precipitation at 37 °C for 20 min. After washed twice, the cell suspension was detected with a FACStar Plus flow cytometer (Becton- Dickinson, USA). The mitochondrial membrane potential could be reflected by the Mean FL2 fluorescence intensity. Annexin V-FITC and PI staining was applied to investigate the cell apoptotic rate by using an Annexin V-FITC Apoptosis Detection Kit. Briefly, after treatment, 500 μL binding buffer was added and then 5 μL Annexin V and 5 μL propidium iodide was loaded in the dark at room temperature for 10 min. Photos were taken under a confocal microscope.
3
Apoptosis Assessment by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were collected from the 6‐well plates and then were incubated with Annexin V‐FITC at a final concentration of 100 ng/mL in the dark for 10 minutes. After washing with PBS, propidium iodide (PI) was used to stain the cells. Flow cytometric analysis was performed for cell apoptosis using a Becton Dickinson FACStar plus flow cytometer according to the instructions of the manual from Becton Dickinson. Data analysis was performed with standard CellQuest software (Becton Dickinson).
4
Annexin V-FITC/PI Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were harvested with trypsin and resuspended in binding buffer. Five microliters Annexin V-fluorescein isothiocyanate and 5 ml propidium iodide (50 mg/ml) were added according to the manufacturer’s protocol (KeyGEN, Nanjing, China). Cells were then analyzed with a FACStar-Plus flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
5
Cellular Uptake of Folate-Conjugated Nanoparticles
6
Annexin V-PI Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurons (106 cells) were collected by centrifugation (2000 g for 10 min at 4 °C) and fixed with 70% ethanol /0.5% Triton X-100. The fixed cells were harvested by centrifugation (2000 g for 10 min at 4 °C) and resuspended in 0.1 ml of PBS containing 500 U/ml RNaseA before they were treated with Annexin V (conjugated with FITC, 5 μl) and Propidium iodide (PI, 10 μl) in the dark at room temperature for 30 min. Cells were sorted by PI and Annexin V by FACs (Becton Dickinson FAC Star Plus flow cytometer).
7
Annexin V-FITC Apoptosis Assay
8
Propidium Iodide Staining of hESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Undifferentiated hESCs were prepared, fixed with 70% ethanol, and stained with 50 μg/ml PI (propidium iodide) as previously described [4 (link)]. Cell cycle distribution (G0–G1, S, and G2/M phases) were determined with flow cytometry (FACStar Plus Flow Cytometer, Becton-Dickinson).
9
Oxidative Stress Evaluation of Carbon Nanotubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5 × 104 V79 or SHE cells were treated with 0.27 to 2.1 μg/cm2 of CNTs for 24 h. Thirty minutes before the end of treatment, 25 μM of 2′,7′-dichlorodihydrofluorescin diacetate (H2DCF-DA, Invitrogen, France) was added to the cultures. Cells were trypsinized and then centrifuged and then placed in HBSS (Hank's buffer saline solution) with 50 μg/mL of propidium iodide. Fluorescence was measured using a Becton Dickinson FACStarPLUS flow cytometer. A sample of nanometric anatase TiO2 [37 (link)] was used for the positive control at 9.2 μg/cm2. Two independent experiments (with duplicates) were realized for every point. Data were expressed as the mean fluorescence intensity of the two experiments ± SD. Statistical analysis was performed using an ANOVA-LSD test (Fisher's least significant difference) (Statgraphics Centurion, Statpoint Technologies, USA).
Potential interference between CNTs and DCF was tested by acellular assays, mixing H2DCF (obtained by NaOH treatment of H2DCF-DA) or DCF fluorescent probe (Sigma-Aldrich, France) and CNTs at different concentrations (from 1 to 250 μg/mL, equivalent to 0.23 to 58 μg/cm2 of cell culture dish). No interference was shown for up to 25 μg/mL (5.8 μg/cm2) of CNTs in SHE cells (data not shown).
Potential interference between CNTs and DCF was tested by acellular assays, mixing H2DCF (obtained by NaOH treatment of H2DCF-DA) or DCF fluorescent probe (Sigma-Aldrich, France) and CNTs at different concentrations (from 1 to 250 μg/mL, equivalent to 0.23 to 58 μg/cm2 of cell culture dish). No interference was shown for up to 25 μg/mL (5.8 μg/cm2) of CNTs in SHE cells (data not shown).
10
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometric analysis of cell cycle was conducted as reported before[28 (link)]. Briefly, adherent and detached HeLa cells were harvested with trypsin, washed with PBS for 3 times and then fixed in ice-cold 70% ethanol at 4°C for 2 h. After centrifugation at 100×g for 2 min, HeLa cells were resuspended in propidium iodide stain buffer (0.1% Triton X-100, 10 μg/mL DNase-free RNase A, 50 μg/mL PI in PBS) for 30 min in dark. Flow cytometric analysis was conducted using a Becton Dickson FACStar Plus Flow Cytometer.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!